TW202034956A - Pharmaceutical compositions comprising anti-191p4d12 antibody drug conjugates and methods of use thereof - Google Patents

Abstract

A pharmaceutical composition comprising an antibody drug conjugate comprising an antibody or antigen binding fragment thereof that binds to 191P4D12 conjugated to one or more units of monomethyl auristatin E (MMAE) and a pharmaceutically acceptable excipient comprising L-histidine, polysorbate-20 (TWEEN-20), and at least one of trehalose dihydrate and sucrose.

Description

Pharmaceutical composition containing anti-191P4D12 antibody drug conjugate and method of use

Provided herein is a pharmaceutical composition comprising an anti-191P4D12 antibody drug conjugate. This article also provides methods of using the pharmaceutical composition.

The drug substance is usually administered in combination with one or more other agents that provide changes and specific medical functions as part of a formulation. Pharmaceutical excipients have various functions and contribute to pharmaceutical formulations in many different ways, such as dissolving, diluting, thickening, stabilizing, preserving, coloring, flavoring, etc. The characteristics that can be considered when formulating the active drug substance include the bioavailability of the dosage form, ease of manufacture, ease of administration, and stability. Due to the varying characteristics of the formulated active drug substance, the dosage form usually requires uniquely customized pharmaceutical excipients for the active drug substance to achieve favorable physical and medical properties.

Therefore, there is a need for pharmaceutical compositions of anti-191P4D12 antibody drug conjugates with favorable physical and medical properties. The present invention meets this need and provides related benefits.

In one aspect, provided herein is a pharmaceutical composition comprising: (a) an antibody-drug conjugate comprising 191P4D12 bound to one or more monomethyl auristatin E (MMAE) units The antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing a complementarity determining region (CDR), the complementarity determining region comprising the heavy chain variable set forth in SEQ ID NO: 7 The amino acid sequence of the CDR of the region; and the light chain variable region containing CDRs, the CDRs including the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 8; and (b) Medicine An academically acceptable excipient comprising at least one of L-histidine, polysorbate-20 (TWEEN-20), and trehalose dihydrate and sucrose.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: CDR H1, which comprises the amino acid sequence of SEQ ID NO: 9; CDR H2, which comprises the amino acid sequence of SEQ ID NO: 10; CDR H3, which Comprises the amino acid sequence of SEQ ID NO: 11; CDR L1, which comprises the amino acid sequence of SEQ ID NO: 12; CDR L2, which comprises the amino acid sequence of SEQ ID NO: 13; and CDR L3, which comprises The amino acid sequence of SEQ ID NO: 14.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the 20th amino acid (glutamate) to the 136th amino acid (seramine) of SEQ ID NO: 7 Acid) within the range of amino acid sequence; and the light chain variable region, which is included in SEQ ID NO: 8 from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) Amino acid sequence within the range.

In some embodiments, the antibody comprises: a heavy chain comprising an amino acid in the range of the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO: 7 Sequence; and light chain, which includes the amino acid sequence in the range of the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO: 8.

In some embodiments, the antigen-binding fragment is a Fab, F(ab') ₂ , Fv or scFv fragment.

In some embodiments, the antibody is a fully human antibody.

In some embodiments, the antibody or antigen-binding fragment thereof is produced recombinantly.

其中L-表示抗體或其抗原結合片段，及p為1至10。In some embodiments, the antibody drug conjugate has the following structure:

Where L- represents an antibody or an antigen-binding fragment thereof, and p is 1-10.

In some embodiments, p is 2-8.

In some embodiments, the antibody or antigen-binding fragment is connected to each monomethyl auristatin E (MMAE) unit via a linker.

In some embodiments, the linker is an enzyme-cleavable linker, and in one embodiment, the linker forms a bond with the sulfur atom of the antibody or antigen-binding fragment thereof.

In some embodiments, the linker has the formula -A _a -W _w -Y _y -; wherein -A- is an extension subunit, a is 0 or 1; -W- is an amino acid unit, and w is at 0 An integer in the range of to 12; and -Y- is a spacer subunit, and y is 0, 1, or 2.

式(1)

式(2)。In some embodiments, the extension subunit has the structure of the following formula (1); the amino acid unit is valine citrulline; and the spacer unit is a PAB group having the structure of the following formula (2):

Formula 1)

Formula (2).

In some embodiments, the extension unit forms a bond with the sulfur atom of the antibody or antigen-binding fragment thereof; and wherein the spacer unit is connected to the MMAE via a carbamate group.

In some embodiments, each antibody or antigen-binding fragment thereof of the antibody-drug conjugate contains 1 to 10 MMAE units.

In some embodiments, each antibody or antigen-binding fragment thereof of the antibody-drug conjugate contains 2 to 8 MMAE units.

In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 1 to about 20 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 5 to about 15 mg/mL. In other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 8 to about 12 mg/mL. In still other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10 mg/mL.

In some embodiments, the content of L-histidine is in the range of about 5 to about 50 mM. In other embodiments, the content of L-histidine is in the range of about 10 to about 40 mM. In other embodiments, the content of L-histidine is in the range of about 15 to about 35 mM. In other embodiments, the content of L-histidine is in the range of about 15 to about 30 mM. In other embodiments, the content of L-histidine is in the range of about 15 to about 25 mM. In still other embodiments, the content of L-histidine is about 20 mM.

In some embodiments, the concentration of TWEEN-20 is in the range of about 0.001 to about 0.1% (v/v). In other embodiments, the concentration of TWEEN-20 is in the range of about 0.0025 to about 0.075% (v/v). In other embodiments, the concentration of TWEEN-20 is in the range of about 0.005 to about 0.05% (v/v). In other embodiments, the concentration of TWEEN-20 is in the range of about 0.01 to about 0.03% (v/v). In still other embodiments, the concentration of TWEEN-20 is in the range of about 0.02% (v/v).

In some embodiments, the pharmaceutical compositions provided herein comprise trehalose dihydrate. In some embodiments, the content of trehalose dihydrate is in the range of about 1 to about 20% (w/v). In some embodiments, the content of trehalose dihydrate is in the range of about 2 to about 15% (w/v). In other embodiments, the content of trehalose dihydrate is in the range of about 3 to about 10% (w/v). In still other embodiments, the content of trehalose dihydrate is in the range of about 4 to about 6% (w/v). In still other embodiments, the content of trehalose dihydrate is about 5.5% (w/v).

In some embodiments, the content of trehalose dihydrate is in the range of about 50 mM to about 300 mM. In some embodiments, the content of trehalose dihydrate is in the range of about 75 mM to about 250 mM. In other embodiments, the content of trehalose dihydrate is in the range of about 100 mM to about 200 mM. In still other embodiments, the content of trehalose dihydrate is in the range of about 130 mM to about 150 mM. In still other embodiments, the content of trehalose dihydrate is about 146 mM.

In some embodiments, the pharmaceutical composition comprises sucrose. In some embodiments, the content of sucrose is in the range of about 1 to about 20% (w/v). In some embodiments, the content of sucrose is in the range of about 2 to about 15% (w/v). In other embodiments, the content of sucrose is in the range of about 3 to about 10% (w/v). In other embodiments, the content of sucrose is in the range of about 4 to about 6% (w/v). In still other embodiments, the content of sucrose is about 5.5% (w/v).

In some embodiments, the content of sucrose is in the range of about 50 mM to about 300 mM. In other embodiments, the sucrose content is in the range of about 75 mM to about 250 mM. In other embodiments, the content of sucrose is in the range of about 100 mM to about 200 mM. In still other embodiments, the content of sucrose is in the range of about 130 mM to about 150 mM. In still other embodiments, the content of sucrose is about 146 mM.

In some embodiments, the pharmaceutical composition has a pH in the range of about 5.5 to about 6.5. In some embodiments, the pharmaceutical composition has a pH in the range of about 5.7 to about 6.3. In other embodiments, the pharmaceutical composition has a pH of about 6.0.

In some embodiments, the pH is measured at room temperature. In some embodiments, the pH is measured at about 15°C to about 27°C. In other embodiments, the pH is measured at about 4°C. In other embodiments, the pH is measured at about 25°C.

In some embodiments, the pharmaceutical compositions provided herein comprise hydrochloric acid (HCl). In some embodiments, the pH is adjusted using HCl.

In other embodiments, the pharmaceutical compositions provided herein comprise succinic acid. In some embodiments, the pH is adjusted using succinic acid.

In some embodiments, the pharmaceutical composition provided herein comprises about 20 mM L-histidine, about 0.02% (w/v) TWEEN-20, and about 5.5% (w/v) trehalose dihydrate or about At least one of 5% (w/v) sucrose. In some embodiments, the pharmaceutical composition provided herein further comprises HCl or succinic acid. In some embodiments, the pH at room temperature is about 6.0. In other embodiments, the pH at 25°C is about 6.0.

其中L-表示抗體或其抗原結合片段，及p為1至10；及(b)醫藥學上可接受之賦形劑，其包含約20 mML-組胺酸、約0.02% (w/v) TWEEN-20、約5.5% (w/v)二水合海藻糖及HCl，其中在25℃下之pH為約6.0。In some specific embodiments, the pharmaceutical composition provided herein includes: (a) an antibody-drug conjugate, which has the following structure:

Wherein L-represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mML-histidine, about 0.02% (w/v) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and HCl, wherein the pH at 25°C is about 6.0.

In some embodiments, the concentration of the antibody drug conjugate is about 10 mg/mL.

其中L-表示抗體或其抗原結合片段，及p為1至10；及 (b)    醫藥學上可接受之賦形劑，其包含約20 mM L-組胺酸、約0.02% (w/v) TWEEN-20、約5.5% (w/v)二水合海藻糖及丁二酸，其中在25℃下之pH為約6.0。In other specific embodiments, the pharmaceutical composition provided herein includes: (a) an antibody-drug conjugate, which includes the following structure:

Wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mM L-histidine, about 0.02% (w/v ) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and succinic acid, wherein the pH at 25°C is about 6.0.

In some embodiments, the concentration of the antibody drug conjugate in the pharmaceutical composition provided herein is about 10 mg/mL.

其中L-表示抗體或其抗原結合片段，及p為1至10；及 (b)    醫藥學上可接受之賦形劑，其包含約20 mM L-組胺酸、約0.02% (w/v) TWEEN-20、約5.0%(w/v)蔗糖及HCl，其中在25℃下之pH為約6.0。In still other specific embodiments, the pharmaceutical composition provided herein includes: (a) an antibody-drug conjugate, which includes the following structure:

Wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mM L-histidine, about 0.02% (w/v ) TWEEN-20, about 5.0% (w/v) sucrose and HCl, wherein the pH at 25°C is about 6.0.

In some embodiments, the concentration of the antibody drug conjugate in the pharmaceutical composition provided herein is about 10 mg/mL.

In some embodiments, the pharmaceutical compositions provided herein are in liquid form.

In other embodiments, the pharmaceutical compositions provided herein are lyophilized.

In another aspect, this document provides a freeze-dried composition, which is prepared by freeze-drying the pharmaceutical composition provided herein.

In some embodiments, the pharmaceutical composition is stored at -80°C, 4°C, 25°C, or 37°C.

In another aspect, provided herein is a method of preventing or treating a disease or condition in an individual, which comprises administering to the individual an effective amount of the pharmaceutical composition provided herein.

In some embodiments, the individual is a human individual.

In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colon cancer, pancreatic cancer, ovarian cancer, lung cancer, bladder cancer, breast cancer, esophageal cancer, head cancer, or neck cancer.

In a specific embodiment, the cancer is colon cancer. In a specific embodiment, the cancer is pancreatic cancer. In a specific embodiment, the cancer is ovarian cancer. In a specific embodiment, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In a specific embodiment, the cancer is bladder cancer. In a specific embodiment, the cancer is advanced bladder cancer. In a specific embodiment, the cancer is metastatic bladder cancer. In a specific embodiment, the cancer is breast cancer. In a specific embodiment, the cancer is esophageal cancer. In a specific embodiment, the cancer is head cancer. In a specific embodiment, the cancer is neck cancer. In a specific embodiment, the cancer has tumor cells expressing 191P4D12.

In some embodiments, the methods provided herein further comprise administering to the individual a second therapeutic agent. In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In other embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In still other embodiments, the PD-1 inhibitor is pembrolizumab or nivolumab. In other embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In other embodiments, the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of 1 to 10 mg/kg body weight. In other embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered at a dose of 1 to 5 mg/kg of the individual's body weight. In still other embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of 1 to 2.5 mg/kg body weight. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of 1 to 1.25 mg/kg body weight. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of about 1 mg/kg body weight. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of about 1.25 mg/kg body weight.

In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion.

In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by two intravenous (IV) injections or infusions for about 30 minutes every three weeks. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion for about 30 minutes on the first and eighth days of every three-week cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion on day 1 of every three-week cycle. In some embodiments, the immune checkpoint inhibitor is pembrolizumab, and pembrolizumab is administered in an amount of about 200 mg over about 30 minutes. In other embodiments, the immune checkpoint inhibitor is atezolizumab, and wherein atezolizumab is administered in an amount of about 1200 mg over about 60 minutes or 30 minutes.

In other embodiments, the antibody drug conjugate formulated in the pharmaceutical composition is administered by three intravenous (IV) injections or infusions for about 30 minutes every four weeks. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion for about 30 minutes on days 1, 8 and 15 of every four-week cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In other embodiments, the immune checkpoint inhibitor is atezolizumab.

Cross reference of related applications

This application claims the priority of U.S. Provisional Patent Application No. 62/774,819 filed on December 3, 2018, the disclosure of which is incorporated herein by reference in its entirety.

Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments set forth herein, and it should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to be limiting. 5.1 Definition

The techniques and procedures described or mentioned in this article include those familiar with the technique using known methods, such as Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd Edition 2001); Current Protocols in Molecular Biology (Ausubel et al., eds. 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering volumes 1 and 2 (Kontermann and Dübel eds., 2nd edition 2010) Describe the widely used methods and generally fully understand and/or commonly use their techniques and procedures.

Unless otherwise defined herein, the technical and scientific terms used in this specification shall have the meanings commonly understood by those who are familiar with the technology. For the purpose of interpreting this specification, the following terminology description will be applied and as long as appropriate, terms used in the singular form will also include the plural form and vice versa. In the event that there is a conflict between any description of the stated terms and any document incorporated herein by reference, the term description set forth below shall prevail.

The terms "antibody", "immunoglobulin" or "Ig" are used interchangeably herein, and are used in the broadest sense and specifically encompass, for example, monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or Complete monoclonal antibodies), antibody compositions with multiple epitopes or single epitope specificities, multiple strains or monovalent antibodies, multivalent antibodies, multispecific antibodies formed by at least two intact antibodies (e.g., bispecific Sex antibodies, as long as they exhibit the required biological activity), single chain antibodies and fragments thereof, as described below. Antibodies can be human antibodies, humanized antibodies, chimeric antibodies and/or affinity mature antibodies, as well as antibodies from other species (such as mice and rabbits). The term "antibody" is intended to include the polypeptide product of the B cell within the polypeptide of the immunoglobulin class, which can bind to a specific molecular antigen and is composed of two pairs of the same polypeptide chain, wherein each pair has a heavy chain (about 50-70 kDa) and a light chain (about 25 kDa), each amino end portion of each chain includes a variable region with about 100 to about 130 or more amino acids, and each carboxyl end portion of each chain includes a constant Area. See, for example, Antibody Engineering (Borrebaeck eds., 2nd edition 1995); and Kuby, Immunology (3rd edition 1997). In specific embodiments, specific molecular antigens can be bound by antibodies (including polypeptides or epitopes) provided herein. Antibodies also include (but are not limited to) synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the above (such as antigen binding Fragments), these functional fragments refer to the part of the antibody heavy chain or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single-chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab) ₂ fragments, F(ab') ₂ fragments, disulfide bond-linked Fv (dsFv), Fd fragments, Fv fragments, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies and minibodies. In particular, the antibodies provided herein include immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, such as antigen binding domains or molecules containing an antigen binding site that binds to an antigen (such as one or more CDRs of an antibody) . Such antibody fragments can be found in, for example, Harlow and Lane, Antibodies: A Laboratory Manual (1989); Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Edited by Myers, 1995); Huston et al., 1993, Cell Biophysics 22:189-224 ; Plückthun and Skerra, 1989, Meth. Enzymol. 178:497-515; and Day, Advanced Immunochemistry (2nd edition 1990). The antibodies provided herein can belong to any class of immunoglobulin molecules (eg, IgG, IgE, IgM, IgD, and IgA) or any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The antibody can be an agonist antibody or an antagonist antibody.

The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies constituting the population are the same except for a small amount of possible naturally occurring mutations. Monoclonal antibodies are highly specific for a single antigen site. In contrast to multiple antibody preparations that can include different antibodies directed against different determinants (epitopes), each monoclonal antibody system is directed against a single determinant on the antigen.

"Antigen" is a structure that an antibody can selectively bind to. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with the cell, such as in the cell, for example, present on or in the cell (e.g., cancer cell).

"Intact" antibodies are antibodies that include an antigen binding site and CL and at least the heavy chain constant regions CH1, CH2, and CH3. The constant region may include a human constant region or amino acid sequence variants thereof. In certain embodiments, intact antibodies have one or more effector functions.

The terms "antigen-binding fragment", "antigen-binding domain", "antigen-binding region" and similar terms refer to antibodies that contain amino acid residues that interact with the antigen and confer the binding agent its specificity for the antigen (e.g., CDR) And the affinity part. As used herein, "antigen-binding fragment" includes "antibody fragments" which comprise a part of a complete antibody, such as the antigen-binding or variable region of the complete antibody. Examples of antibody fragments include (but are not limited to) Fab, Fab', F(ab') ₂ and Fv fragments; diabodies and di-diabodies (see, for example, Holliger et al., 1993, Proc. Natl. Acad. Sci 90:6444-48; Lu et al., 2005, J. Biol. Chem. 280:19665-72; Hudson et al., 2003, Nat. Med. 9:129-34; WO 93/11161; and U.S. Patent No. No. 5,837,242 and No. 6,492,123); Single-chain antibody molecules (see, for example, U.S. Patent Nos. 4,946,778; No. 5,260,203; No. 5,482,858; and No. 5,476,786); dual variable domain antibodies (see, for example, U.S. Patent No. 7,612,181); Single variable domain antibody (sdAb) (see, for example, Woolven et al., 1999, Immunogenetics 50: 98-101; and Streltsov et al., 2004, Proc Natl Acad Sci USA. 101: 12444-49); and those formed from antibody fragments Multispecific antibodies.

The term "binds/binding" refers to the interaction between molecules, including, for example, the formation of complexes. The interactions can be, for example, non-covalent interactions, including hydrogen bonds, ionic bonds, hydrophobic interactions and/or van der Waals interactions. Complexes can also include the combination of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The total non-covalent interaction strength between a single antigen binding site on an antibody and a single epitope of a target molecule (such as an antigen) is the affinity of the antibody or functional fragment for the epitope. The ratio (k _off / _kon ) of the dissociation rate (k _off ) to the binding rate (k _on ) of a binding molecule (such as an antibody) and a monovalent antigen (k _off / _kon ) is the dissociation constant K _D , which is negatively related to the affinity. The lower the K _D value, the higher the antibody affinity. The K _D value varies with different complexes of antibody and antigen and depends _on k _on and k _off . The dissociation constant K _D of the antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at a binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple and repeated antigenic determinants (such as a multivalent antigen) is in contact with an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site will increase at the second site The probability of response. The strength of such multiple interactions between a multivalent antibody and an antigen is called avidity.

In conjunction with the antibodies or antigen-binding fragments thereof described herein, terms such as "bind to", "that specifically bind to" and similar terms may also be used herein Used interchangeably, and refers to a binding molecule that has an antigen-binding domain that specifically binds to an antigen (such as a polypeptide). Antibodies or antigen-binding fragments that bind to or specifically bind to antigens can cross-react with related antigens. In some embodiments, the antibody or antigen-binding fragment that binds to or specifically binds to the antigen does not cross-react with other antigens. Antibodies or antigen-binding fragments that bind to an antigen or specifically bind to an antigen can be identified, for example, by immunoassay, Octet ^® , Biacore ^® or other techniques known to those skilled in the art. In some embodiments, when the antibody or antigen-binding fragment binds to the antigen with a higher affinity than any cross-reacting antigen, such as using experimental techniques (such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA)) As determined, the antibody or antigen-binding fragment binds to the antigen or specifically binds to the antigen. Generally, the specific or selective reaction will be at least twice the background signal or noise and can be greater than 10 times the background. For a discussion of binding specificity, see, for example, Fundamental Immunology 332-36 (Edited by Paul, 2nd edition, 1989). In some embodiments, the degree of binding of the antibody or antigen-binding fragment to the "non-target" protein is less than about 10% of the binding of the binding molecule or antigen-binding domain to its specific target antigen, for example, by fluorescent activation of cell sorting ( FACS) analysis or RIA determination. Terms such as "specific binding", "specifically binds to" or "is specific for" mean that it is measurably different from The combination of non-specific interactions. Specific binding can be measured, for example, by comparing the binding of the measuring molecule with the binding of a control molecule, which is generally a molecule with a similar structure without binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target (e.g., excess unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled targets, specific binding is indicated. Antibodies or antigen-binding fragments that bind to antigen include antibodies or antigen-binding fragments that are capable of binding to the antigen with sufficient affinity to make the binding molecule useful as, for example, a diagnostic agent in the targeted antigen. In certain embodiments, the antibody or antigen-binding fragment that binds to the antigen has a dissociation constant (K _D ) less than or equal to: 1000 nM, 800 nM, 500 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, the antibody or antigen-binding fragment binds to an epitope of an antigen that is conserved among antigens from different species (for example, between humans and cynomolgus monkey species).

"Binding affinity" generally refers to the total strength of the non-covalent interaction between a single binding site of a molecule (eg, a binding protein, such as an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of the binding molecule X to its binding partner Y can generally be represented by the dissociation constant (K _D ). Affinity can be measured by common methods known in the art (including those described herein). Low-affinity antibodies generally bind antigen slowly and tend to be easily broken down, while high-affinity antibodies generally bind antigen faster and tend to remain bound for a longer time. Various methods for measuring binding affinity are known in the art, any of which can be used for the purpose of the present invention. Specific illustrative embodiments include the following. In one embodiment, "K _D "or "K _D value" can be measured by analysis known in the art, for example, by combined analysis. K _D can be measured by RIA, for example, using the Fab format of the antibody of interest and its antigen (Chen et al., 1999, J. Mol Biol 293:865-81). K _D or K _D values can also be analyzed by using biological layer interferometry (BLI) or surface plasmon resonance (SPR) by Octet®, using, for example, Octet® QK384 system, or by Biacore®, using, for example, Biacore® TM-2000 or Biacore®TM-3000 for measurement. "On-rate/rate of association/association rate" or "kon" can also use the same biological layer interferometry (BLI) or surface plasmon resonance (SPR) techniques described above, using, for example, Octet® QK384, Biacore®TM-2000 or Biacore®TM-3000 system to determine.

In certain embodiments, the antibody or antigen-binding fragment may comprise a "chimeric" sequence, where a part of the heavy chain and/or light chain is identical to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass Or it is homologous, and the rest of the chain is identical to or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass and fragments of such antibodies, as long as it presents The required biological activity is sufficient (see US Patent No. 4,816,567; and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-55).

In certain embodiments, antibodies or antigen-binding fragments may comprise portions of "humanized" forms of non-human (e.g., murine) antibodies, which are chimeric antibodies (e.g., receptors) that include human immunoglobulins. Antibody), wherein the native CDR residues are derived from the corresponding CDR of a non-human species (such as mouse, rat, rabbit, or non-human primate) (such as a donor antibody) with the required specificity, affinity, and ability Residue replacement. In some cases, one or more FR region residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues not found in the recipient antibody or the donor antibody. These modifications are made to further optimize antibody performance. The humanized antibody heavy chain or light chain may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to those of non-human immunoglobulins, and all or substantially all of the FRs are human immunoglobulins FR of protein sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), usually at least a portion of a human immunoglobulin. For other details, see Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1988, Nature 332:323-29; Presta, 1992, Curr. Op. Struct. Biol. 2:593-96; Carter Et al., 1992, Proc. Natl. Acad. Sci. USA 89: 4285-89; U.S. Patent No. 6,800,738; No. 6,719,971; No. 6,639,055; No. 6,407,213; and No. 6,054,297.

In certain embodiments, the antibody or antigen-binding fragment may comprise part of a "fully human antibody" or "human antibody", where these terms are used interchangeably herein and refer to include human variable regions and, for example, human constant regions The antibody. In certain embodiments, these terms refer to antibodies comprising human variable and constant regions. In certain embodiments, "fully human" antibodies can also encompass antibodies that bind to polypeptides and are encoded by nucleic acid sequences that are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequences. The term "fully human antibody" includes antibodies containing variable and constant regions corresponding to human germline immunoglobulin sequences as described by Kabat et al. (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest , p. Fifth edition, US Department of Health and Human Services, NIH Publication No. 91-3242). "Human antibodies" are antibodies whose amino acid sequences correspond to those of antibodies produced by humans and/or have been prepared using any technology for preparing human antibodies. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using various techniques known in this technology, including phage display libraries (Hoogenboom and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581 ) And yeast presentation library (Chao et al., 2006, Nature Protocols 1: 755-68). Cole et al., Monoclonal Antibodies and Cancer Therapy 77 (1985); Boerner et al., 1991, J. Immunol. 147(1):86-95; and van Dijk and van de Winkel, 2001, Curr. Opin. Pharmacol. 5 : The method described in 368-74 can also be used to prepare human monoclonal antibodies. Human antibodies can be prepared by administering antigens to transgenic animals (e.g. mice) whose endogenous loci have been modified to produce such antibodies in response to antigen stimulation (see e.g. Jakobovits, 1995, Curr. Opin. Biotechnol. 6(5): 561-66; Brüggemann and Taussing, 1997, Curr. Opin. Biotechnol. 8(4): 455-58; and U.S. Patent Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE ^TM technology). For human antibodies produced via human B-cell fusion tumor technology, see also, for example, Li et al., 2006, Proc. Natl. Acad. Sci. USA, 103:3557-62.

In certain embodiments, the antibody or antigen-binding fragment may comprise part of a "recombinant human antibody", where the phrase includes a human antibody prepared, expressed, produced, or isolated by recombinant means, such as the use of transfection into host cells The antibody expressed by the recombinant expression vector; the antibody isolated from the recombinant combinatorial human antibody library; the antibody isolated from the transgenic human immunoglobulin gene and/or the transgenic animal (such as mouse or cow) (see for example Taylor, LD et al. (1992) Nucl. Acids Res. 20:6287-6295) or by any other means involving the splicing of human immunoglobulin gene sequences to other DNA sequences to prepare, express, produce or isolate antibodies. Such recombinant human antibodies may have variable and constant regions derived from human germline immunoglobulin sequences (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). However, in certain embodiments, such recombinant human antibodies are subject to in vitro mutagenesis (or somatic mutagenesis in vivo when animal transgenic genes of human Ig sequences are used), and therefore the VH and VL of recombinant antibodies Although the amino acid sequence of the region is derived from and related to the human germline VH and VL sequences, it does not naturally exist in the human germline antibody library in vivo.

In certain embodiments, an antibody or antigen-binding fragment may comprise part of a "monoclonal antibody", where as used herein, the term refers to a population of substantially homogeneous antibodies (e.g., except for possible naturally occurring In addition to the mutation, the individual antibodies that make up the population are identical) obtained antibodies, and each monoclonal antibody will usually recognize a single epitope on the antigen. In a specific embodiment, as used herein, a "monoclonal antibody" is an antibody produced by a single fusion tumor or other cell. The term "monoclonal" is not limited to any specific method used to prepare antibodies. For example, monoclonal antibodies suitable for use in the present invention can be prepared by the fusion tumor method described by Kohler et al., 1975, Nature 256:495 for the first time, or recombinant DNA can be used in bacterial or eukaryotic animal or plant cells Method preparation (see, for example, U.S. Patent No. 4,816,567). The "monoclonal antibody" can also use the technique described in Clackson et al., 1991, Nature 352:624-28 and Marks et al., 1991, J. Mol. Biol., 222:581-97, autophagosome antibody library In the separation. Other methods for preparing pure cell lines and monoclonal antibodies expressed by them are well known in the art. See, for example, Short Protocols in Molecular Biology (Ausubel et al. eds., 5th edition. 2002).

A typical 4-chain antibody unit is a heterotetraglycan protein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is generally about 150,000 Daltons. Each L chain is connected to the H chain via a covalent disulfide bond, and the two H chains are connected to each other via one or more disulfide bonds depending on the same type of the H chain. Each H chain and L chain also have intrachain disulfide bridges that are regularly separated. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) (for each of the α and γ chains) and four CH domains (for the μ and ε isotypes) Words). Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. Align VL with VH, and align CL with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains. VH and VL pair together to form a single antigen binding site. For the structure and characteristics of different classes of antibodies, see, for example, Basic and Clinical Immunology 71 (Stites et al., 8th edition 1994); and Immunobiology (Janeway et al., 5th edition 2001).

The term "Fab" or "Fab region" refers to the region of an antibody that binds to an antigen. Conventional IgG usually contains two Fab regions, each residing on one of the two arms of the Y-shaped IgG structure. Each Fab region usually consists of a variable region and a constant region of each of the heavy chain and the light chain. More specifically, the variable and constant regions of the heavy chain in the Fab region are the VH and CH1 regions, and the variable and constant regions of the light chain in the Fab region are the VL and CL regions. The VH, CH1, VL, and CL in the Fab region can be arranged in various ways to confer antigen binding ability according to the present invention. For example, the VH and CH1 regions can be on one polypeptide, and the VL and CL regions can be on separate polypeptides, similar to the Fab region of conventional IgG. Alternatively, the VH, CH1, VL, and CL regions can all be on the same polypeptide and oriented in a different order as described in more detail in the following sections.

The term "variable region", "variable domain", "V region" or "V domain" refers to a part of the light or heavy chain of an antibody, which is usually located at the amino end of the light or heavy chain and in the heavy chain The medium length is about 120 to 130 amino acids and the length in the light chain is about 100 to 110 amino acids, and is used for the binding and specificity of each specific antibody to its specific antigen. The variable region of the heavy chain can be referred to as "VH". The variable region of the light chain can be referred to as "VL". The term "variable" refers to the fact that certain segments of the variable region differ widely in sequence in the antibody. The V region mediates antigen binding and defines the specificity of a specific antibody against its specific antigen. However, the variability is unevenly distributed within the 110 amino acid spans of the variable region. In fact, the V domain consists of the following: weakly variable (e.g., relatively constant) fragments of about 15-30 amino acids (called framework regions (FR)), which are shorter and more variable (e.g., Regions of extreme variability (called "hypervariable regions", each about 9-12 amino acids long) are separated. The variable regions of the heavy chain and the light chain each contain four FRs that largely adopt a β-sheet configuration. These FRs are connected by three hypervariable regions that form a loop connecting the β-sheet structure and are In some cases, it forms part of the beta sheet structure. The hypervariable regions in each chain are tightly combined with the hypervariable regions from other chains by FR to promote the formation of antibody antigen binding sites (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest (5th Edition, 1991). )). The constant region is not directly involved in the binding of antibodies and antigens, but exhibits a variety of effector functions, such as involving antibodies in antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The variable regions differ widely in sequence between different antibodies. In a specific embodiment, the variable region is a human variable region.

The term "numbering according to the variable region residues of Kabat" or "numbering of amino acid positions as in Kabat" and its variants refer to Kabat et al., see above for the variable region or light chain of the heavy chain of the antibody. The numbering system of the variable area. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 (residue 52a, according to Kabat) and three insertion residues after residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). For a given antibody, the Kabat numbering of residues can be determined by aligning the homology regions of the antibody sequence with the "standard" Kabat numbering sequence. The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in the constant region of an immunoglobulin heavy chain (for example, Kabat et al., see EU index reported above). "EU index in Kabat" refers to the residue number of human IgG1 EU antibody. Other numbering systems have been described by, for example, AbM, Chothia, Contact, IMGT, and AHon.

When used with reference to an antibody, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino end portion includes a variable region having about 120 to 130 or more amino acids, and the carboxyl end portion Including the constant region. Based on the amino acid sequence of the constant region of the heavy chain, the constant region can be one of five different types (such as the same type), called alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (µ). Different heavy chains have different sizes: α, δ, and γ contain approximately 450 amino acids, while µ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains produce five well-known classes (such as isotypes) of antibodies, namely IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG, namely IgG1, IgG2, and IgG3 and IgG4.

When used with reference to an antibody, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino terminal portion includes a variable region having about 100 to about 110 or more amino acids, and the carboxy terminal portion includes Constant region. The approximate length of the light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, called kappa (κ) or lambda (λ).

As used herein, the terms "hypervariable region", "HVR", "complementarity determining region" and "CDR" are used interchangeably. "CDR" refers to one of the three hypervariable regions (H1, H2 or H3) in the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or the non-framework of the antibody VL β-sheet framework One of the three hypervariable areas (L1, L2 or L3) in the framework area. Therefore, CDR is a variable region sequence interspersed within the framework region sequence.

The CDR regions are well known to those skilled in the art and have been defined by a well-known numbering system. For example, the Kabat complementarity determining region (CDR) is based on sequence variability and is most commonly used (see, for example, Kabat et al., supra). Chothia relates to the position of structural loops (see, for example, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). When using the Kabat numbering convention for numbering, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion in H35A and H35B; if neither exists If 35A and 35B are not present, the end of the ring is at 32; if only 35A is present, the end of the ring is at 33; if both 35A and 35B are present, the end of the ring is at 34). The AbM hypervariable region represents the balance between the Kabat CDR and the Chothia structural loop, and is used by Oxford Molecular's AbM antibody modeling software (see, for example, Antibody Engineering Vol. 2 (Kontermann and Dübel eds, 2nd edition, 2010)). The "contact" hypervariable zone is based on the analysis of available composite crystal structures. Another universal numbering system that has been developed and widely adopted is ImmunoGeneTics (IMGT) Information ^System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system dedicated to immunoglobulin (IG), T cell receptor (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates. Herein, CDRs are mentioned with regard to the amino acid sequence and the position in the light chain or heavy chain. Since the "position" of the CDR in the immunoglobulin variable domain structure is conserved between species and exists in a structure called a loop, it is easy to identify the CDR using a numbering system that aligns the variable domain sequence according to structural features And framework residues. This information can be used to transplant and replace CDR residues from immunoglobulins from a species into the acceptor framework typically derived from human antibodies. Honegger and Plückthun, 2001, J. Mol. Biol. 309: 657-70 have developed another numbering system (AHon). The correspondence between numbering systems (including, for example, Kabat numbering and IMGT unique numbering systems) is well known to those skilled in the art (see, for example, Kabat, see above; Chothia and Lesk, see above; Martin, see above; Lefranc Et al., see above). The residues from each of these hypervariable regions or CDRs are indicated below. Table 30 Kabat AbM Chothia Contact IMGT CDR L1 L24--L34 L24--L34 L24--L34 L30--L36 L27--L38 CDR L2 L50--L56 L50--L56 L50--L56 L46--L55 L56--L65 CDR L3 L89--L97 L89--L97 L89--L97 L89--L96 L105-L117 CDR H1 H31--H35B H26--H35B H26--H32..34 H30--H35B H27--H38 (Kabat number) CDR H1 H31--H35 H26--H35 H26--H32 H30--H35 (Chothia number) CDR H2 H50--H65 H50--H58 H52--H56 H47--H58 H56--H65 CDR H3 H95--H102 H95--H102 H95--H102 H93--H101 H105-H117

The boundaries of the established CDR can vary depending on the identification scheme. Therefore, unless otherwise specified, the terms "CDR" and "complementarity determining region" or regions of a given antibody, such as variable regions, and individual CDRs of antibodies (eg CDR-H1, CDR-H2) or regions thereof, should be understood as Covers the complementary determining region as defined by any of the known solutions described above. In some cases, a scheme for identifying one or more specific CDRs is specified, such as CDRs defined by Kabat, Chothia, or Contact methods. In other cases, the specific amino acid sequence of the CDR is given.

Hypervariable regions can include the following "extended hypervariable regions": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL; And 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in VH.

The term "constant region" or "constant domain" refers to the carboxy-terminal part of the light chain and the heavy chain, which is not directly involved in the binding of an antibody to an antigen, but exhibits multiple effector functions, such as interaction with Fc receptors. The term refers to the part of an immunoglobulin molecule that contains amino acid sequences that are more conserved than other parts of the immunoglobulin (variable regions, which contain antigen binding sites). The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

The term "framework" or "FR" refers to the variable region residues flanking the CDRs. FR residues are present in, for example, chimeric antibodies, humanized antibodies, human antibodies, domain antibodies, bifunctional antibodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than hypervariable region residues or CDR residues.

As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc region, recombinant Fc region and variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is usually defined as an amino acid residue at position Cys226 or extending from Pro230 to its carboxy terminus. The C-terminal lysine (residue 447, according to the EU numbering system) of the Fc region can be removed, for example, during the preparation or purification of the antibody, or by recombinantly engineering the nucleic acid encoding the heavy chain of the antibody. Therefore, the composition of intact antibodies may include an antibody population in which all K447 residues have been removed, an antibody population in which K447 residues have not been removed, and an antibody population containing a mixture of antibodies with and without K447 residues. The "functional Fc region" has the "effect function" of the native sequence Fc region. Exemplary "effector functions" include C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down-regulation of cell surface receptors (such as B cell receptors) and the like. Such effector functions usually require the combination of an Fc region and a binding region or a binding domain (such as an antibody variable region or domain), and can be assessed using various analytical methods known to those skilled in the art. The "variant Fc region" includes an amino acid sequence that differs from the native sequence Fc region by at least one amino acid modification (for example, substitution, addition, or deletion). In certain embodiments, the variant Fc region has at least one amino acid in the native sequence Fc region or in the Fc region of the parent polypeptide compared to the native sequence Fc region or compared to the Fc region of the parent polypeptide. Substitution, for example, about one to about ten amino acid substitutions, or about one to about five amino acid substitutions. The variant Fc region herein may have at least about 80% homology with the native sequence Fc region and/or the Fc region of the parent polypeptide, or at least about 90% homology with it, for example, at least about 95% homology with it. Source.

As used herein, "antigenic determinant" is a term in the art and refers to the localized region of an antigen to which a binding molecule (such as an antibody) can specifically bind. The epitope can be a linear epitope or a conformational, non-linear or discontinuous epitope. For example, in the case of a polypeptide antigen, the epitope can be a contiguous amino acid of the polypeptide ("linear" epitope), or the epitope can include two or more non-contiguous regions of the polypeptide. Amino acids ("configurational", "non-linear" or "discontinuous" epitopes). Those familiar with this technology should understand that, in general, linear epitopes may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, the binding molecule binds to a set of amino acids, regardless of whether it folds in the natural three-dimensional protein structure. In other embodiments, the binding molecule requires the amino acid residues constituting the epitope to exhibit a specific configuration (for example, bending, twisting, turning or folding) in order to recognize and bind the epitope.

The terms "polypeptide" and "peptide" and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. These terms also encompass amino acid polymers that have been naturally modified or modified by intervention (such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification). This definition also includes, for example, polypeptides containing one or more amino acid analogs (including but not limited to non-natural amino acids) and other modifications known in the art. It should be understood that since the polypeptides of the present invention may be based on antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the "polypeptide" may appear in the form of a single chain or two or more related chains.

The term "vector" refers to a substance used to carry or include a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a binding molecule (eg, an antibody) as described herein to introduce the nucleic acid sequence into a host cell. Suitable vectors include, for example, expression vectors, plastids, phage vectors, viral vectors, episomes and artificial chromosomes, which may include selection sequences or markers operable to stably integrate into the host cell chromosome. In addition, the vector may include one or more selectable marker genes and appropriate performance control sequences. The selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiency, or supply key nutrients that are not in the medium. Performance control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators and the like well known in the art. When two or more nucleic acid molecules are co-expressed (for example, antibody heavy and light chains or antibody VH and VL), both nucleic acid molecules can be inserted into, for example, a single expression vector or a separate expression vector. For a single vector expression, the encoding nucleic acid can be operatively linked to a common expression control sequence, or to different expression control sequences (such as an inducible promoter and a constitutive promoter). The introduction of nucleic acid molecules into host cells can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as the Northern blot method or polymerase chain reaction (PCR) amplification of mRNA; or the immunoblotting method for gene product expression, or testing the performance of the introduced nucleic acid sequence or its corresponding gene product Other suitable analytical methods. Those familiar with the art should understand that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product, and it should also be understood that the expressed amount can be optimized using methods well known in the art to obtain sufficient performance.

As used herein, the term "host" refers to animals, such as mammals (e.g., humans).

As used herein, the term "host cell" refers to a specific individual cell that can be transfected with nucleic acid molecules and the progeny or potential progeny of such cells. Due to the possibility of mutations or environmental influences or the integration of nucleic acid molecules into the host cell genome in the progeny, the progeny of such cells may be inconsistent with the parent cells transfected with nucleic acid molecules.

"Isolated nucleic acid" is a nucleic acid, such as RNA, DNA, or hybrid nucleic acid, which is substantially separated from other genomic DNA sequences and proteins or complexes (such as ribosomes and polymerases) that naturally accompany native sequences. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. In addition, "isolated" nucleic acid molecules (such as cDNA molecules) may be substantially free of other cellular materials or culture media (when prepared by recombinant technology), or substantially free of chemical precursors or other chemical substances (when chemically synthesized Time). In a specific embodiment, one or more nucleic acid molecules encoding antibodies as described herein are isolated or purified. The term encompasses nucleic acid sequences that have been removed from a naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biosynthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule.

As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs or can be incorporated into the polymerization by DNA or RNA polymerase or by synthesis reactions Any substance in the matter. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, "oligonucleotide" refers to a short, usually single-stranded synthetic polynucleotide, which is usually (but not necessarily) less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. The cells that produce the binding molecules of the present invention may include parental fusion tumor cells, as well as bacterial and eukaryotic host cells into which the nucleic acid encoding the antibody has been introduced. Unless otherwise specified, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5'end; the left-hand direction of a double-stranded polynucleotide sequence is called the 5'direction. The 5'to 3'direction of addition of the nascent RNA transcript is called the transcription direction; the DNA strand contains the same sequence as the RNA transcript, and the sequence region 5'to the 5'end of the RNA transcript is called the "upstream sequence"; DNA The region of the strand that contains the same sequence as the RNA transcript and is 3'to the 3'end of the RNA transcript is called the "downstream sequence".

As used herein, the term "pharmaceutically acceptable" means approved by the regulatory agency of the federal or state government or listed in the US Pharmacopia ( US Pharmacopia ), European Pharmacopia ( European Pharmacopia ) or other recognized pharmacopoeia for use in animals, and more Human beings in specific terms.

"Excipient" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material. Excipients include, for example, encapsulating materials or additives, such as absorption accelerators, antioxidants, binders, buffers, carriers, coating agents, coloring agents, diluents, disintegrants, emulsifiers, extenders, fillers Agents, flavoring agents, humectants, lubricants, fragrances, preservatives, propellants, release agents, sterilizing agents, sweeteners, solubilizers, wetting agents and mixtures thereof. The term "excipient" can also refer to diluents, adjuvants (for example Freunds' adjuvant (complete or incomplete)) or vehicles.

In some embodiments, the excipient is a pharmaceutically acceptable excipient. Examples of pharmaceutically acceptable excipients include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (for example, less than about 10 amino acid residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as l-histidine, glycine, glutamic acid, asparagine, Arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, sucrose, trehalose dihydrate, mannose or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol ; Salt-forming relative ions, such as sodium; and/or non-ionic surfactants, such as TWEEN™, polyethylene glycol (PEG) and PLURONICS™. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, Remington's Pharmaceutical Sciences (18th edition 1990).

In one embodiment, each component is "pharmaceutically acceptable" in the following sense: it is compatible with other ingredients of the pharmaceutical formulation and is suitable for use in contact with human and animal tissues or organs without excessive toxicity, Irritation, allergic reaction, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio. See, for example, Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th edition; Rowe et al. Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd edition; Ash and Ash Edited; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd edition; Gibson edited; CRC Press LLC: Boca Raton, FL, 2009. In some embodiments, the pharmaceutically acceptable excipient is non-toxic to cells or mammals exposed to it at the dosage and concentration used. In some embodiments, the pharmaceutically acceptable excipient is an aqueous pH buffered solution.

In some embodiments, the excipients are sterile liquids, such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the composition (e.g., pharmaceutical composition) is administered intravenously, water is an exemplary excipient. Saline solution and aqueous dextrose and glycerol solutions can also be used as liquid excipients, especially for injectable solutions. Excipients can also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicone, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, Propylene, glycol, water, ethanol and the like. If necessary, the composition may also contain a small amount of wetting or emulsifying agent, or pH buffering agent. The composition can be in the form of a solution, suspension, emulsion, lozenge, pill, capsule, powder, sustained release formulation and the like.

Compositions (including pharmaceutical compounds) may contain binding molecules (such as antibodies), for example in isolated or purified form, together with suitable amounts of excipients.

The abbreviation "MMAE" refers to monomethyl auristatin E.

Unless otherwise indicated, the term "alkyl" refers to a saturated straight or branched chain hydrocarbon containing about 1 to about 20 carbon atoms (and all range combinations and subcombinations and specific numbers of carbon atoms therein), of which about 1 Up to about 8 carbon atoms are preferred. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, second butyl, tertiary butyl, n-pentyl, 2-pentyl, 3-pentyl , 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl Group, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl and 3,3-dimethyl-2-butyl. Alkyl groups (either alone or as part of another group) may optionally be substituted with one or more groups (preferably 1 to 3 groups) (and any additional substituents selected from halogen), and such substitutions Groups include (but are not limited to) -halogen, -O-(C ₁ -C ₈ alkyl), -O- (C ₂ -C ₈ alkenyl), -O- (C ₂ -C ₈ alkynyl), -Aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N (R') ₂ , -NHC(O)R', -SR', -SO ₃ R', -S(O) ₂ R', -S(O)R', -OH, =O, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ and -CN, wherein each R'is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ Alkenyl, -C ₂ -C ₈ alkynyl or -aryl, and these -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl), -O-( C ₂ -C ₈ alkynyl), -aryl, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl and -C ₂ -C ₈ alkynyl may further include (but not limited to) the following as appropriate One or more group substitutions: -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -halogen, -O-(C ₁ -C ₈ alkyl ), -O-(C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH ₂ , -C(O)NHR'', -C(O)N(R'') ₂ , -NHC(O)R'', -SR '', -SO ₃ R'', -S(O) ₂ R'', -S(O)R'', -OH, -N ₃ , -NH ₂ , -NH(R''), -N (R") ₂ and -CN, wherein each R" is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl or -Aryl.

Unless otherwise indicated, the terms "alkenyl" and "alkynyl" refer to straight and branched carbon chains containing about 2 to about 20 carbon atoms (and all range combinations and subcombinations and specific numbers of carbon atoms therein) Among them, about 2 to about 8 carbon atoms are preferred. The alkenyl chain has at least one double bond in the chain, and the alkynyl chain has at least one triple bond in the chain. Examples of alkenyl include (but are not limited to) ethylene or vinyl, allyl,-1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl Alkenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl and -2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but are not limited to, acetylenic, propargyl, ethynyl, propynyl, -1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl and -3-methyl-1-butynyl. Alkenyl and alkynyl groups (whether alone or as part of another group) may optionally be substituted with one or more groups (preferably 1 to 3 groups) (and any additional substituents selected from halogen), Such substituent groups include (but are not limited to) -halogen, -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkyne基), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C( O)N(R') ₂ , -NHC(O)R', -SR', -SO ₃ R', -S(O) ₂ R', -S(O)R', -OH, =O, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ and -CN, wherein each R'is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, or -aryl, and among them, -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl),- O-(C ₂ -C ₈ alkynyl), -aryl, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl and -C ₂ -C ₈ alkynyl may be further included (but not Limited to) one or more of the following substituents: -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -halogen, -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl), -aryl, -C(O)R'', -OC(O)R '', -C(O)OR'', -C(O)NH ₂ , -C(O)NHR'', -C(O)N(R'') ₂ , -NHC(O)R'' , -SR'', -SO ₃ R'', -S(O) ₂ R'', -S(O)R'', -OH, -N ₃ , -NH ₂ , -NH(R'') , -N(R'') ₂ and -CN, wherein each R'' is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ Alkynyl or -aryl.

Unless otherwise indicated, the term "alkylene" refers to a carbon atom containing from about 1 to about 20 (and all range combinations and subcombinations and a specific number of carbon atoms therein) (wherein from about 1 to about 8 carbon atoms are more Preferably) and has a saturated branched or straight chain hydrocarbon group with two monovalent group centers obtained by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane. Typical alkylene groups include (but are not limited to) methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylylene, decylene, 1,4-cyclohexylene and the like. The alkylene group (whether alone or as part of another group) may optionally be substituted with one or more groups (preferably 1 to 3 groups) (and any additional substituents selected from halogen), which Substituent groups include (but are not limited to) -halogen, -O-(C ₁ -C ₈ alkyl), -O- (C ₂ -C ₈ alkenyl), -O- (C ₂ -C ₈ alkynyl) , -Aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O) N(R') ₂ , -NHC(O)R', -SR', -SO ₃ R', -S(O) ₂ R', -S(O)R', -OH, =O, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ and -CN, wherein each R'is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl or -aryl, and among these -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl), -O- (C ₂ -C ₈ alkynyl), -aryl, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl and -C ₂ -C ₈ alkynyl may further include (but not limited to) ) One or more of the following substituents are substituted: -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -halogen, -O-(C ₁ -C ₈ Alkyl), -O-(C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl), -aryl, -C(O)R'', -OC(O)R'',-C(O)OR'', -C(O)NH ₂ , -C(O)NHR'', -C(O)N(R'') ₂ , -NHC(O)R'', -SR'', -SO ₃ R'', -S(O) ₂ R'', -S(O)R'', -OH, -N ₃ , -NH ₂ , -NH(R''), -N(R'') ₂ and -CN, wherein each R'' is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkyne基 or -aryl.

Unless otherwise indicated, the term "alkenylene" refers to an optionally substituted alkylene group containing at least one carbon-carbon double bond. Exemplary alkenylene groups include, for example, vinylene (-CH=CH-) and propenylene (-CH=CHCH ₂ -).

Unless otherwise indicated, the term "alkynylene" refers to optionally substituted alkylene groups containing at least one carbon-carbon triple bond. Exemplary alkynylene groups include, for example, ethynylene (-C≡C-), propynylene (-CH ₂ C≡C-) and 4-pentynyl (-CH ₂ CH ₂ CH ₂ C≡CH-) .

Unless otherwise indicated, the term "aryl" refers to a group having 6-20 carbon atoms (and all range combinations and sub-combinations and specific numbers thereof) obtained by removing one hydrogen atom from a single carbon atom of the parent aromatic ring system Carbon atoms) monovalent aromatic hydrocarbon group. Some aryl groups are represented as "Ar" in the exemplary structure. Typical aryl groups include, but are not limited to, groups derived from benzene, substituted benzene, phenyl, naphthalene, anthracene, biphenyl and the like.

The aryl group (either alone or as part of another group) may optionally be substituted with one or more, preferably 1 to 5 or even 1 to 2 groups, and these substituent groups include (but are not limited to) -Halogen, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -O-(C ₁ -C ₈ alkyl), -O-(C _2- C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C( O)NH ₂ , -C(O)NHR', -C(O)N(R') ₂ , -NHC(O)R', -SR', -SO ₃ R', -S(O) ₂ R ', -S(O)R', -OH, -NO ₂ , -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ and -CN, wherein each R'is independently Selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl or -aryl, and among these -C ₁ -C ₈ alkyl,- C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl) and-aryl may be further optionally substituted with one or more substituents including (but not limited to) the following: -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -halogen, -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl), -aryl基, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH ₂ , -C(O)NHR'', -C(O )N(R'') ₂ , -NHC(O)R'', -SR'', -SO ₃ R'', -S(O) ₂ R'', -S(O)R'',- OH, -N ₃ , -NH ₂ , -NH(R''), -N(R'') ₂ and -CN, wherein each R'' is independently selected from -H, -C ₁ -C ₈ alkane Group, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl or -aryl.

， 典型的「-(C₁ -C₈ 伸烷基)芳基」、「-(C₂ -C₈ 伸烯基)芳基」及「-(C₂ -C₈ 伸炔基)芳基」包括(但不限於)苯甲基、2-苯基乙-1-基、2-苯基伸乙-1-基、萘基甲基、2-萘基乙-1-基、2-萘基伸乙-1-基、萘并苯甲基、2-萘并苯基乙-1-基及其類似者。Unless otherwise specified, the term "aryl" refers to optionally substituted aryl groups that are divalent (ie, by removing two hydrogens from the same or two different carbon atoms of the parent aromatic ring system). Atoms are derived) and can be in ortho, occasional, or pair configurations, as shown in the structure below, with phenyl as an exemplary aryl group.

, Typical "-(C ₁ -C ₈ alkylene) aryl", "-(C ₂ -C ₈ alkenylene) aryl" and "-(C ₂ -C ₈ alkynylene) aryl" Including (but not limited to) benzyl, 2-phenylethyl-1-yl, 2-phenylethylene-1-yl, naphthylmethyl, 2-naphthylethyl-1-yl, 2-naphthylethylene-1-yl -1-yl, naphthobenzyl, 2-naphthophenylethyl-1-yl and the like.

啶基、嗎啉基、噁唑啶基、苯并三唑基、苯并異噁唑基、羥吲哚基、苯并噁唑啉基及靛紅醯基。較佳的「雜環」基團包括(但不限於)苯并呋喃基、苯并噻吩基、吲哚基、苯并吡唑基、香豆素基、異喹啉基、吡咯基、噻吩基、呋喃基、噻唑基、咪唑基、吡唑基、三唑基、喹啉基、嘧啶基、吡啶基、吡啶酮基、吡嗪基、噠嗪基、異噻唑基、異噁唑基及四唑基。雜環基團(無論單獨還是作為另一基團之一部分)可視情況經一或多個基團，較佳地1至2個基團取代，該等取代基團包括(但不限於)-C₁ -C₈ 烷基、-C₂ -C₈ 烯基、-C₂ -C₈ 炔基、-鹵素、-O-(C₁ -C₈ 烷基)、-O-(C₂ -C₈ 烯基)、-O-(C₂ -C₈ 炔基)、-芳基、-C(O)R'、-OC(O)R'、-C(O)OR'、-C(O)NH₂ 、-C(O)NHR'、-C(O)N(R')₂ 、-NHC(O)R'、-SR'、-SO₃ R'、-S(O)₂ R'、-S(O)R'、-OH、-N₃ 、-NH₂ 、-NH(R')、-N(R')₂ 及-CN，其中各R'係獨立地選自-H、-C₁ -C₈ 烷基、-C₂ -C₈ 烯基、-C₂ -C₈ 炔基或-芳基，且其中該等-O-(C₁ -C₈ 烷基)、-O-(C₂ -C₈ 烯基)、-O-(C₂ -C₈ 炔基)、-C₁ -C₈ 烷基、-C₂ -C₈ 烯基、-C₂ -C₈ 炔基及-芳基可進一步視情況經包括(但不限於)以下之一或多個取代基取代：-C₁ -C₈ 烷基、-C₂ -C₈ 烯基、-C₂ -C₈ 炔基、-鹵素、-O-(C₁ -C₈ 烷基)、-O-(C₂ -C₈ 烯基)、-O-(C₂ -C₈ 炔基)、-芳基、-C(O)R''、-OC(O)R''、-C(O)OR''、-C(O)NH₂ 、-C(O)NHR''、-C(O)N(R'')₂ 、-NHC(O)R''、-SR''、-SO₃ R''、-S(O)₂ R''、-S(O)R''、-OH、-N₃ 、-NH₂ 、-NH(R'')、-N(R'')₂ 及-CN，其中各R''係獨立地選自-H、-C₁ -C₈ 烷基、-C₂ -C₈ 烯基、-C₂ -C₈ 炔基或芳基。Unless otherwise indicated, the term "heterocyclic ring" refers to a monocyclic, bicyclic or polycyclic ring system with 3 to 14 ring atoms (also called ring members), wherein at least one ring atom in at least one ring is selected from Heteroatoms of N, O, P, or S (and all range combinations and sub-combinations and specific numbers of carbon atoms and heteroatoms). The heterocyclic ring may have 1 to 4 ring heteroatoms independently selected from N, O, P, or S. One or more of the N, C, or S atoms in the heterocycle may be oxidized. The monocyclic heterocycle preferably has 3 to 7 ring members (for example, 2 to 6 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P or S), and the bicyclic heterocycle preferably It has 5 to 10 ring members (for example, 4 to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N, O, P, or S). The ring including the heteroatom may be an aromatic ring or a non-aromatic ring. Unless otherwise indicated, the heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Heterocycles are described in Paquette, Leo A., " Principles of Modern Heterocyclic Chemistry " (WA Benjamin, New York, 1968), specifically Chapters 1, 3, 4, 6, 7 and 9; " The Chemistry of Heterocyclic Compounds , A series of Monographs " (John Wiley & Sons, New York, 1950 to present), specifically Volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. 82:5566 (1960) . Examples of "heterocyclic" groups include (by way of example and not limitation) pyridinyl, dihydropyridinyl, tetrahydropyridinyl (piperidinyl), thiazolyl, pyrimidinyl, furyl, thienyl, pyrrolyl, pyridine Azolyl, imidazolyl, tetrazolyl, benzofuranyl, thionaphthyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidine Keto, pyrrolidinyl, 2-pyrrolidinone, pyrrolinyl, tetrahydrofuranyl, bistetrahydrofuranyl, tetrahydropiperanyl, bistetrahydropiperanyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl , Decahydroquinolinyl, octahydroisoquinolinyl, azepine, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl , Thienyl, thienyl, piperanyl, isobenzofuranyl, pyranyl, pyrrolyl, phenoxathiyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazine Group, indolazinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinazinyl, phthalazinyl, pyridinyl, quinolinyl, quinazolinyl , Phenanthinyl, pteridine, 4H-carbazolyl, carbazolyl, β-carboline, phenanthridinyl, acridinyl, pyrimidinyl, phenantholinyl, phenazidinyl, phenanthiazinyl, furfur Xyl, phenoxazinyl, iso-alkyl, pyrazolidinyl, imidazolinyl, pyrazolinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl,

Ridinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindole, benzoxazolinyl and isatinyl. Preferred "heterocyclic" groups include (but are not limited to) benzofuranyl, benzothienyl, indolyl, benzopyrazolyl, coumarin, isoquinolinyl, pyrrolyl, thienyl , Furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl and tetrazolium Azole. The heterocyclic group (either alone or as part of another group) may optionally be substituted with one or more groups, preferably 1 to 2 groups, and such substituent groups include (but are not limited to) -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -halogen, -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ Alkenyl), -O-(C ₂ -C ₈ alkynyl), -aryl, -C(O)R', -OC(O)R', -C(O)OR', -C(O) NH ₂ , -C(O)NHR', -C(O)N(R') ₂ , -NHC(O)R', -SR', -SO ₃ R', -S(O) ₂ R', -S(O)R', -OH, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ and -CN, wherein each R'is independently selected from -H,- C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl or -aryl, and wherein these -O-(C ₁ -C ₈ alkyl), -O- (C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl), -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl and -The aryl group may be further optionally substituted by one or more substituents including (but not limited to) the following: -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl , -Halogen, -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl), -O- (C ₂ -C ₈ alkynyl), -aryl, -C( O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH ₂ , -C(O)NHR'', -C(O)N(R'') ₂ , -NHC(O)R'', -SR'', -SO ₃ R'', -S(O) ₂ R'', -S(O)R'', -OH, -N ₃ , -NH ₂ , -NH(R''), -N(R'') ₂ and -CN, wherein each R'' is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl or aryl.

As an example and not limitation, a carbon-bonded heterocycle can be bonded at the following positions: position 2, 3, 4, 5 or 6 of pyridine; position 3, 4, 5 or 6 of pyridazine; position of pyrimidine 2, 4, 5 or 6 positions; 2, 3, 5, or 6 positions of pyrazine; 2, 3, 4, or 5 positions of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole; oxazole, Position 2, 4 or 5 of imidazole or thiazole; position 3, 4 or 5 of isoxazole, pyrazole or isothiazole; position 2 or 3 of aziridine; position 2, 3 of azetidine Or 4 positions; 2, 3, 4, 5, 6, 7 or 8 positions for quinoline; or 1, 3, 4, 5, 6, 7 or 8 positions for isoquinoline. More generally, carbon-bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridyl Azinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrimidinyl Azinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen-bonded heterocycles can be bonded at the following positions: aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazoline, 1 position of 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline or 1H-indazole ; 2 positions for isoindole or isoindoline; 4 positions for morpholine; and 9 positions for carbazole or β-carboline. More generally, nitrogen-bonded heterocycles include 1-aziridine, 1-azetidinyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

Unless otherwise indicated, the term "carbocyclic ring" refers to a saturated or unsaturated non-aromatic monocyclic, bicyclic or polycyclic ring with 3 to 14 ring atoms (and all range combinations and subcombinations and specific numbers of carbon atoms therein) A ring system in which all ring atoms are carbon atoms. The monocyclic carbocyclic ring preferably has 3 to 6 ring atoms, and more preferably 5 or 6 ring atoms. The bicyclic carbocyclic ring preferably has 7 to 12 ring atoms, for example arranged in a bicyclic [4,5], [5,5], [5,6] or [6,6] system; or 9 or 10 ring atoms , The arrangement is a double ring [5,6] or [6,6] system. The term "carbocyclic ring" includes, for example, a monocyclic carbocyclic ring fused with an aromatic ring (e.g., a monocyclic carbocyclic ring fused with a benzene ring). The carbocyclic ring preferably has 3 to 8 carbon ring atoms. The carbocyclic group (whether alone or as part of another group) may optionally be substituted with, for example, one or more groups, preferably 1 or 2 groups (and any additional substituents selected from halogen), the Such substituent groups include (but are not limited to) -halogen, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -O-(C ₁ -C ₈ alkane基), -O-(C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl), -aryl, -C(O)R', -OC(O)R',- C(O)OR', -C(O)NH ₂ , -C(O)NHR', -C(O)N(R') ₂ , -NHC(O)R', -SR', -SO ₃ R', -S(O) ₂ R', -S(O)R', -OH, =O, -N ₃ , -NH ₂ , -NH(R'), -N(R') ₂ and- CN, wherein each R'is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl or -aryl, and wherein these- C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl) ), -O-(C ₂ -C ₈ alkynyl) and -aryl may be further substituted with one or more substituents including (but not limited to) the following as appropriate: -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, -halogen, -O-(C ₁ -C ₈ alkyl), -O-(C ₂ -C ₈ alkenyl), -O-(C ₂ -C ₈ alkynyl), -aryl, -C(O)R'', -OC(O)R'', -C(O)OR'', -C(O)NH ₂ , -C( O)NHR'', -C(O)N(R'') ₂ , -NHC(O)R'', -SR'', -SO ₃ R'', -S(O) ₂ R'', -S(O)R'', -OH, -N ₃ , -NH ₂ , -NH(R''), -N(R'') ₂ and -CN, wherein each R'' is independently selected from -H, -C ₁ -C ₈ alkyl, -C ₂ -C ₈ alkenyl, -C ₂ -C ₈ alkynyl, or -aryl.

Examples of monocyclic carbocyclic substituents include -cyclopropyl, -cyclobutyl, -cyclopentyl, -1-cyclopent-1-enyl, -1-cyclopent-2-enyl, -1-cyclo Pent-3-enyl, cyclohexyl,-1-cyclohex-1-enyl,-1-cyclohex-2-enyl,-1-cyclohex-3-enyl, -cycloheptyl, -cyclo Octyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl and- Cyclooctadienyl.

"Carbocyclic ring" (whether used alone or as part of another group) refers to a divalent (ie, by removing the same or two different carbon atoms from the parent carbocyclic ring system) optionally substituted It is a carbocyclic group as defined above.

Unless the context dictates otherwise, the hyphen (-) indicates the point of attachment to the side molecule. Therefore, the term "-(C ₁ -C ₈ alkylene) aryl" or "-C ₁ -C ₈ alkylene (aryl)" refers to C ₁ -C ₈ alkylene as defined herein, Wherein the alkylene is connected to the pendant molecule at any one of the carbon atoms of the alkylene, and one of the hydrogen atoms bonded to the carbon atom of the alkylene is replaced by an aryl group as defined herein.

When a specific group is "substituted", the group may have one or more substituents, preferably one to five substituents, more preferably one to three substituents, and most preferably one to two substituents, such substituents Independently selected from the list of substituents. However, the group can generally have any number of substituents selected from halogens. This indicates substituted groups. It is hoped that the definition of any substituent or change at a particular position in the molecule is independent of its definition at other positions in the molecule. It should be understood that the substituents and substitution patterns of the compounds of the present invention can be selected by those skilled in the art to provide compounds that are chemically stable and can be easily synthesized by techniques known in the art and their methods described herein.

The protecting group as used herein refers to a group that selectively blocks (temporarily or permanently) a reactive site in a polyfunctional compound. Suitable hydroxy-protecting groups for use in the present invention are pharmaceutically acceptable, and for the compound to be active, may or may not be cleaved from the parent compound after administration to the individual. Lysis is through the normal metabolic process in the body. Hydroxy protecting groups are well known in the art, see Protective Groups in Organic Synthesis (John Wiley & sons, 3rd edition) by TW Greene and PGM Wuts (which is incorporated herein by reference in its entirety and for all purposes ), and includes, for example, ethers (such as alkyl ethers; and silyl ethers, including, for example, dialkylsilyl ethers, trialkylsilyl ethers, dialkyl alkoxysilyl ethers), esters, carbonates, and amines Protective groups for methyl formate, sulfonate and phosphate. Examples of hydroxyl protecting groups include (but are not limited to) methyl ether; methoxymethyl ether, methylthiomethyl ether, (phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl Base ether, p-methoxybenzyloxy methyl ether, p-nitrobenzyloxy methyl ether, o-nitrobenzyloxy methyl ether, (4-methoxyphenoxy) methyl ether , O-methoxyphenol methyl ether, tertiary butoxy methyl ether, 4-pentenoxy methyl ether, silyloxy methyl ether, 2-methoxyethoxy methyl ether, 2, 2,2-Trichloroethoxy methyl ether, bis (2-chloroethoxy) methyl ether, 2-(trimethylsilyl) ethoxy methyl ether, methoxy methyl ether, four Hydropiperanyl ether, 1-methoxycyclohexyl ether, 4-methoxytetrahydropyranyl ether, 4-methoxytetrahydropyranyl ether S,S-dioxide, 1- [(2-Chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether, 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl ether , 1,4-dioxan-2-yl ether, tetrahydrofuranyl ether, tetrahydrosulfuryl ether; substituted ethyl ether (such as 1-ethoxyethyl ether, 1-(2-chloroethoxy Base) ethyl ether, 1-[2-(trimethylsilyl)ethoxy]ethyl ether, 1-methyl-1-methoxyethyl ether, 1-methyl-1-benzyloxy Ethyl ether, 1-methyl-1-benzyloxy-2-fluoroethyl ether, 1-methyl-1-phenoxyethyl ether), 2-trimethylsilyl ether, third Butyl ether, allyl ether, propargyl ether, p-chlorophenyl ether, p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether, 3,4-dimethoxybenzene Methyl ether, trimethylsilyl ether, triethylsilyl ether, tripropylsilyl ether, dimethylisopropylsilyl ether, diethylisopropylsilyl ether, dimethylhexylsilyl Base ether, tertiary butyl dimethyl silyl ether, diphenyl methyl silyl ether, benzyl formate, acetate, chloroacetate, dichloroacetate, trichloroacetate , Trifluoroacetate, Methoxyacetate, Triphenylmethoxyacetate, Phenylacetate, Benzoate, Alkyl Carbonate Methyl, Alkyl Carbonate 9-Methyl Ester , Ethyl alkyl carbonate, 2,2,2,-trichloroethyl carbonate, 1,1,-dimethyl-2,2,2-trichloroethyl carbonate, alkyl sulfonate , Methane sulfonate, benzyl sulfonate, toluene sulfonate, methylene acetal, ethylene acetal and tertiary butyl methylene ketal. The preferred protecting group is represented by the following formula: -R ^a , -Si(R ^a )(R ^a )(R ^a ), -C(O)R ^a , -C(O)OR ^a , -C(O) NH(R ^a ), -S(O) ₂ R ^a , -S(O) ₂ OH, P(O)(OH) ₂ and -P(O)(OH)OR ^a , where R ^a is C _1- C ₂₀ alkyl, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl, -C ₁ -C ₂₀ alkylene (carbocyclic), -C ₂ -C ₂₀ alkenylene (carbocyclic),- C ₂ -C ₂₀ alkynylene (carbocyclic), -C ₆ -C ₁₀ aryl, -C ₁ -C ₂₀ alkylene (aryl), -C ₂ -C ₂₀ alkenylene (aryl), -C ₂ -C ₂₀ alkynylene (aryl), -C ₁ -C ₂₀ alkylene (heterocyclic), -C ₂ -C ₂₀ alkenylene (heterocyclic) or -C ₂ -C ₂₀ alkynylene Group (heterocyclic), in which the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic and heterocyclic groups (either alone or as part of another group) Part) Replaced as appropriate.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a binding molecule (eg, antibody) or pharmaceutical composition provided herein that is sufficient to produce the desired result.

The terms "individual" and "patient" are used interchangeably. As used herein, in certain embodiments, the individual is a mammal, such as a non-primate animal (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate animal (e.g., monkey and human). In a specific embodiment, the individual is a human. In one embodiment, the individual is a mammal diagnosed with a condition or disorder, such as a human. In another embodiment, the individual is a mammal, such as a human, who is at risk of developing a condition or disorder.

"Administer/administration" refers to the operation of injecting or otherwise physically delivering substances present in the body into the patient's body, such as by transmucosal, intradermal, intravenous, intramuscular delivery, and/or as described herein Or any other physical delivery method known in the art.

As used herein, the term "treat/treatment/treating" refers to a reduction or improvement in the progression, severity, and/or duration of a disease or condition resulting from the administration of one or more therapies. Treatment can be determined by assessing whether there has been a reduction, alleviation, and/or alleviation of one or more symptoms associated with the underlying condition, so that improvement in the patient is observed, although the patient may still suffer from the underlying condition. The term "treatment" includes both controlling and improving disease. The term "manage/managing/management" refers to the beneficial effects obtained by the individual from the therapy, which may not cause the cure of the disease.

The term "prevent/preventing/prevention" refers to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or related symptoms (such as cancer).

The term "cancer" or "cancer cell" is used herein to refer to tissues or cells found in neoplasms, which have characteristics that distinguish them from normal tissues or tissue cells. Such characteristics include (but are not limited to): degree of degeneration, irregular shape, unclear cell outline, nuclear size, changes in nuclear or cytoplasmic structure, other phenotypic changes, cells that indicate cancerous or precancerous states The presence of protein increases the number of mitosis and the ability to metastasize. The term "cancer" includes cancer, sarcoma, tumor, epithelioma, leukemia, lymphoma, polyp and sclerocarcinoma, transformation, neoplasm and the like.

The term "about" or "approximately" means within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, 4% of a predetermined value or range Within, within 3%, within 2%, 1% or less.

Unless the context clearly dictates otherwise, as used in the present invention and the scope of the patent application, the singular forms "一 (a/an)" and "the (the)" include plural forms.

It should be understood that whenever the term "comprising" is used herein to describe an embodiment, other similar embodiments described with the terms "consisting of" and/or "essentially consisting of" are also provided. It should also be understood that whenever the phrase "substantially consists of" is used in this document to describe an embodiment, another similar embodiment described with the term "consisting of" is also provided.

The term "and/or" as used in phrases such as "A and/or B" herein is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B, and/or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A Or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). 5.2 Pharmaceutical composition

In one aspect, a "pharmaceutical composition" is provided herein, which includes the antibody drug conjugate provided herein and one or more pharmaceutically acceptable or physiologically acceptable excipients. In certain embodiments, the antibody drug conjugate is provided in combination with one or more additional agents or separately therefrom. A composition is also provided, which comprises such one or more additional agents and one or more pharmaceutically acceptable or physiologically acceptable excipients. In certain embodiments, the antibody drug conjugate and the additional agent are present in therapeutically acceptable amounts. The pharmaceutical composition can be used according to the methods and uses provided herein. Therefore, for example, the pharmaceutical composition can be administered to an individual in vitro or in vivo in order to practice the treatment methods and uses provided herein. The pharmaceutical composition of the present invention can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are described herein.

In some embodiments, there are provided pharmaceutical compositions of antibody-drug conjugates that modulate cancer or tumors.

In some aspects, the pharmaceutical composition may further include other therapeutically active agents or compounds as disclosed herein or known to those skilled in the art to be useful in the treatment or prevention of various diseases and disorders (such as cancer) as described herein. As explained above, additional therapeutically active agents or compounds may be present in separate pharmaceutical compositions.

The pharmaceutical composition generally comprises a therapeutically effective amount of at least one of the antibody-drug conjugates provided herein and one or more pharmaceutically acceptable formulation agents. In certain embodiments, the pharmaceutical composition further comprises one or more additional agents described herein.

In one embodiment, the pharmaceutical composition comprises the antibody drug conjugate provided herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of the antibody drug conjugate provided herein. In certain embodiments, the pharmaceutical composition includes pharmaceutically acceptable excipients.

In some embodiments, the antibody-drug conjugates in the pharmaceutical compositions provided herein are selected from the antibody-drug conjugates described in Section 5.3 below.

In certain embodiments, the pharmaceutical composition comprises an antibody drug conjugate at a concentration of 0.1-100 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of 1 to 20 mg/mL. In other embodiments, the pharmaceutical composition comprises an antibody drug conjugate at a concentration of 5 to 15 mg/mL. In other embodiments, the pharmaceutical composition comprises an antibody drug conjugate at a concentration of 8 to 12 mg/mL. In other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of 9 to 11 mg/mL. In some embodiments, the pharmaceutical composition comprises an antibody drug conjugate at a concentration of about 9.5 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.6 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.7 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.8 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 9.9 mg/mL. In still other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10 mg/mL. In still other embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.1 mg/mL. In some embodiments, the pharmaceutical composition comprises an antibody drug conjugate at a concentration of about 10.2 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.3 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.3 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.4 mg/mL. In some embodiments, the pharmaceutical composition comprises the antibody drug conjugate at a concentration of about 10.5 mg/mL.

In some embodiments, the pharmaceutical composition provided herein includes at least one of L-histidine, TWEEN-20, and trehalose dihydrate or sucrose. In some embodiments, the pharmaceutical composition provided herein further comprises hydrochloric acid (HCl) or succinic acid.

In some embodiments, the concentration of L-histidine suitable for use in the pharmaceutical compositions provided herein is in the range of 5 to 50 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is in the range of 10 to 40 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is in the range of 15 to 35 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is in the range of 15 to 30 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is in the range of 15 to 25 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is in the range of 15 to 35 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 16 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 17 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 18 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 19 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 20 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 21 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 22 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 23 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 24 mM. In some embodiments, the concentration of L-histidine in the pharmaceutical compositions provided herein is about 25 mM.

In some embodiments, the concentration of TWEEN-20 suitable for use in the pharmaceutical compositions provided herein is in the range of 0.001 to 0.1% (v/v). In another embodiment, the concentration of TWEEN-20 is in the range of 0.0025 to 0.075% (v/v). In one embodiment, the concentration of TWEEN-20 is in the range of 0.005 to 0.05% (v/v). In another embodiment, the concentration of TWEEN-20 is in the range of 0.0075 to 0.025% (v/v). In another embodiment, the concentration of TWEEN-20 is in the range of 0.0075 to 0.05% (v/v). In another embodiment, the concentration of TWEEN-20 is in the range of 0.01 to 0.03% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.01% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.015% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.016% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.017% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.018% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.019% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.02% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.021% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.022% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.023% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.024% (v/v). In a specific embodiment, the concentration of TWEEN-20 is about 0.025% (v/v).

In one embodiment, the concentration of trehalose dihydrate suitable for use in the pharmaceutical composition provided herein is in the range of 1% to 20% (w/v). In another embodiment, the concentration of trehalose dihydrate is in the range of 2% to 15% (w/v). In one embodiment, the concentration of trehalose dihydrate is in the range of 3% to 10% (w/v). In another embodiment, the concentration of trehalose dihydrate is in the range of 4% to 9% (w/v). In another embodiment, the concentration of trehalose dihydrate is in the range of 4% to 8% (w/v). In another embodiment, the concentration of trehalose dihydrate is in the range of 4% to 7% (w/v). In another embodiment, the concentration of trehalose dihydrate is in the range of 4% to 6% (w/v). In another embodiment, the concentration of trehalose dihydrate is in the range of 4.5% to 6% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 4.6% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 4.7% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 4.8% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 4.9% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.0% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.1% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.2% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.3% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.4% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.5% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.6% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.7% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.8% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 5.9% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 6.0% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 6.1% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 6.2% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 6.3% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 6.4% (w/v). In another embodiment, the concentration of trehalose dihydrate is about 6.5% (w/v).

In certain embodiments, the molar concentration of trehalose dihydrate is 50 to 300 mM. In other embodiments, the molar concentration of trehalose dihydrate is 75 to 250 mM. In some embodiments, the molar concentration of trehalose dihydrate is 100 to 200 mM. In other embodiments, the molar concentration of trehalose dihydrate is 130 to 150 mM. In some embodiments, the molar concentration of trehalose dihydrate is 135 to 150 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 135 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 136 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 137 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 138 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 139 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 140 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 141 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 142 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 143 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 144 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 145 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 146 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 150 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 151 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 151 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 152 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 153 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 154 mM. In certain embodiments, the molar concentration of trehalose dihydrate is about 155 mM.

In one embodiment, the concentration of sucrose suitable for use in the pharmaceutical compositions provided herein is in the range of 1% to 20% (w/v). In another embodiment, the concentration of sucrose is in the range of 2% to 15% (w/v). In one embodiment, the concentration of sucrose is in the range of 3% to 10% (w/v). In another embodiment, the concentration of sucrose is in the range of 4% to 9% (w/v). In another embodiment, the concentration of sucrose is in the range of 4% to 8% (w/v). In another embodiment, the concentration of sucrose is in the range of 4% to 7% (w/v). In another embodiment, the concentration of sucrose is in the range of 4% to 6% (w/v). In another embodiment, the concentration of sucrose is in the range of 4.5% to 6% (w/v). In another embodiment, the concentration of sucrose is about 4.6% (w/v). In another embodiment, the concentration of sucrose is about 4.7% (w/v). In another embodiment, the concentration of sucrose is about 4.8% (w/v). In another embodiment, the concentration of sucrose is about 4.9% (w/v). In another embodiment, the concentration of sucrose is about 5.0% (w/v). In another embodiment, the concentration of sucrose is about 5.1% (w/v). In another embodiment, the concentration of sucrose is about 5.2% (w/v). In another embodiment, the concentration of sucrose is about 5.3% (w/v). In another embodiment, the concentration of sucrose is about 5.4% (w/v). In another embodiment, the concentration of sucrose is about 5.5% (w/v). In another embodiment, the concentration of sucrose is about 5.6% (w/v). In another embodiment, the concentration of sucrose is about 5.7% (w/v). In another embodiment, the concentration of sucrose is about 5.8% (w/v). In another embodiment, the concentration of sucrose is about 5.9% (w/v). In another embodiment, the concentration of sucrose is about 6.0% (w/v). In another embodiment, the concentration of sucrose is about 6.1% (w/v). In another embodiment, the concentration of sucrose is about 6.2% (w/v). In another embodiment, the concentration of sucrose is about 6.3% (w/v). In another embodiment, the concentration of sucrose is about 6.4% (w/v). In another embodiment, the concentration of sucrose is about 6.5% (w/v).

In certain embodiments, the molar concentration of sucrose is 50 to 300 mM. In other embodiments, the molar concentration of sucrose is 75 to 250 mM. In some embodiments, the molar concentration of sucrose is 100 to 200 mM. In other embodiments, the molar concentration of sucrose is 130 to 150 mM. In some embodiments, the molar concentration of sucrose is 135 to 150 mM. In certain embodiments, the molar concentration of sucrose is about 135 mM. In certain embodiments, the molar concentration of sucrose is about 136 mM. In certain embodiments, the molar concentration of sucrose is about 137 mM. In certain embodiments, the molar concentration of sucrose is about 138 mM. In certain embodiments, the molar concentration of sucrose is about 139 mM. In certain embodiments, the molar concentration of sucrose is about 140 mM. In certain embodiments, the molar concentration of sucrose is about 141 mM. In certain embodiments, the molar concentration of sucrose is about 142 mM. In certain embodiments, the molar concentration of sucrose is about 143 mM. In certain embodiments, the molar concentration of sucrose is about 144 mM. In certain embodiments, the molar concentration of sucrose is about 145 mM. In certain embodiments, the molar concentration of sucrose is about 146 mM. In certain embodiments, the molar concentration of sucrose is about 150 mM. In certain embodiments, the molar concentration of sucrose is about 151 mM. In certain embodiments, the molar concentration of sucrose is about 151 mM. In certain embodiments, the molar concentration of sucrose is about 152 mM. In certain embodiments, the molar concentration of sucrose is about 153 mM. In certain embodiments, the molar concentration of sucrose is about 154 mM. In certain embodiments, the molar concentration of sucrose is about 155 mM.

In some embodiments, the pharmaceutical compositions provided herein comprise HCl. In other embodiments, the pharmaceutical compositions provided herein comprise succinic acid.

In some embodiments, the pharmaceutical compositions provided herein have a pH in the range of 5.5 to 6.5. In other embodiments, the pharmaceutical composition provided herein has a pH in the range of 5.7 to 6.3. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.7. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.8. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 5.9. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.0. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.1. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.2. In some embodiments, the pharmaceutical compositions provided herein have a pH of about 6.3.

In some embodiments, the pH is measured at room temperature. In other embodiments, the pH is measured at 15°C to 27°C. In still other embodiments, the pH is measured at 4°C. In still other embodiments, the pH is measured at 25°C.

In some embodiments, the pH is adjusted using HCl. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.5 to 6.5 at room temperature. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.7 to 6.3 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.7 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.8 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.9 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.0 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.1 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.2 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.3 at room temperature.

In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.5 to 6.5 at 15°C to 27°C. In some embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH in the range of 5.7 to 6.3 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.2 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises HCl, and the pharmaceutical composition has a pH of about 6.3 at 15°C to 27°C.

In some embodiments, the pH is adjusted using succinic acid. In some embodiments, the pharmaceutical composition includes succinic acid, and the pharmaceutical composition has a pH in the range of 5.5 to 6.5 at room temperature. In some embodiments, the pharmaceutical composition includes succinic acid, and the pharmaceutical composition has a pH in the range of 5.7 to 6.3 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.7 at room temperature. In some more specific embodiments, the pharmaceutical composition includes succinic acid, and the pharmaceutical composition has a pH of about 5.8 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.9 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.0 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.1 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.2 at room temperature. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.3 at room temperature.

In some embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH in the range of 5.5 to 6.5 at 15°C to 27°C. In some embodiments, the pharmaceutical composition includes succinic acid, and the pharmaceutical composition has a pH in the range of 5.7 to 6.3 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.7 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.8 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 5.9 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.0 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.1 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.2 at 15°C to 27°C. In some more specific embodiments, the pharmaceutical composition comprises succinic acid, and the pharmaceutical composition has a pH of about 6.3 at 15°C to 27°C.

In some specific embodiments, the pharmaceutical composition provided herein comprises about 20 mM L-histidine, about 0.02% (w/v) TWEEN-20, and about 5.5% (w/v) trehalose dihydrate or about At least one of 5% (w/v) sucrose. In some embodiments, the pharmaceutical composition provided herein further comprises HCl or succinic acid. In some embodiments, the pH at room temperature is about 6.0. In some embodiments, the pH at 25°C is about 6.0.

In some specific embodiments, the pharmaceutical composition provided herein comprises about 20 mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and HCl . In some embodiments, the pH at room temperature is about 6.0. In some embodiments, the pH at 25°C is about 6.0.

In some specific embodiments, the pharmaceutical composition provided herein comprises about 20 mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5% (w/v) sucrose, and HCl. In some embodiments, the pH at room temperature is about 6.0. In some embodiments, the pH at 25°C is about 6.0.

In other specific embodiments, the pharmaceutical composition provided herein comprises about 20 mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and butyl Diacid. In some embodiments, the pH at room temperature is about 6.0. In some embodiments, the pH at 25°C is about 6.0.

In some specific embodiments, the pharmaceutical compositions provided herein comprise about 20 mM L-histidine, about 0.02% (w/v) TWEEN-20, about 5% (w/v) sucrose, and succinic acid. In some embodiments, the pH at room temperature is about 6.0. In some embodiments, the pH at 25°C is about 6.0.

其中L-表示抗體或其抗原結合片段，及p為1至10；及 (b)    醫藥學上可接受之賦形劑，其包含約20 mM L-組胺酸、約0.02% (w/v) TWEEN-20、約5.5%(w/v)二水合海藻糖及HCl，其中抗體藥物結合物之濃度為約10 mg/mL，且其中在25℃下之pH為約6.0。In a specific embodiment, the (a) antibody-drug conjugate provided herein includes the following structure:

Wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mM L-histidine, about 0.02% (w/v ) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and HCl, wherein the concentration of the antibody-drug conjugate is about 10 mg/mL, and the pH at 25°C is about 6.0.

其中L-表示抗體或其抗原結合片段，及p為1至10；及 (b)    醫藥學上可接受之賦形劑，其包含約20 mM L-組胺酸、約0.02% (w/v) TWEEN-20、約5.5% (w/v)二水合海藻糖及丁二酸， 其中抗體藥物結合物之濃度為約10 mg/mL，且其中在25℃下之pH為約6.0。In another specific embodiment, the pharmaceutical composition provided herein comprises: (a) an antibody-drug conjugate comprising the following structure:

Wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mM L-histidine, about 0.02% (w/v ) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and succinic acid, wherein the concentration of the antibody-drug conjugate is about 10 mg/mL, and the pH at 25°C is about 6.0.

其中L-表示抗體或其抗原結合片段，及p為1至10；及 (b)    醫藥學上可接受之賦形劑，其包含約20 mM L-組胺酸、約0.02% (w/v) TWEEN-20、約5.0% (w/v)蔗糖及HCl， 其中抗體藥物結合物之濃度為約10 mg/mL，且其中在25℃下之pH為約6.0。In yet another specific embodiment, the pharmaceutical composition provided herein comprises: (a) an antibody-drug conjugate, which comprises the following structure:

Wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mM L-histidine, about 0.02% (w/v ) TWEEN-20, about 5.0% (w/v) sucrose and HCl, wherein the concentration of the antibody-drug conjugate is about 10 mg/mL, and the pH at 25°C is about 6.0.

Although some numbers (and their numerical ranges) are provided, it should be understood that, in some embodiments, they are also covered within, for example, 2%, 5%, 10%, 15%, or 20% of the number (or numerical range) The numerical value. Other exemplary pharmaceutical compositions are provided in the experimental section below.

The main solvent in the vehicle can be aqueous or non-aqueous in nature. In addition, the vehicle may contain other pharmaceutically acceptable excipients for modifying or maintaining the pH, osmolality, viscosity, sterility, or stability of the pharmaceutical composition. In certain embodiments, the pharmaceutically acceptable vehicle is an aqueous buffer. In other embodiments, the vehicle includes, for example, sodium chloride and/or sodium citrate.

The pharmaceutical compositions provided herein may also contain other pharmaceutically acceptable formulation agents for modifying or maintaining the release rate of the antibody drug conjugates and/or additional agents as described herein. Such formulated agents include those substances known to those skilled in the preparation of sustained-release formulations. For other references regarding pharmaceutical and physiologically acceptable formulations, see, for example, Remington's Pharmaceutical Sciences , 18th Edition (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, The Merck Index , 12th edition (1996, Merck Publishing Group, Whitehouse, NJ); and Pharmaceutical Principles of Solid Dosage Forms (1993, Technonic Publishing Co., Inc., Lancaster, Pa.). Additional pharmaceutical compositions suitable for administration are known in the art and can be applied to the methods and compositions provided herein.

In some embodiments, the pharmaceutical compositions provided herein are in liquid form. In other embodiments, the pharmaceutical composition provided herein has been lyophilized.

The pharmaceutical composition can be stored in a sterile bottle in the form of a solution, suspension, gel, emulsion, solid or dehydrated or lyophilized powder. Such compositions can be stored in a ready-to-use form, a lyophilized form that needs to be reconstituted before use, a liquid form that needs to be diluted before use, or other acceptable forms. In some embodiments, the pharmaceutical composition is provided in a single-use container (e.g., single-use vial, ampoule, syringe, or auto-injector (similar to, for example, EpiPen®)), while in other embodiments, it is provided in multiple-use containers. In a container (e.g. multiple use vial). Any drug delivery device can be used to deliver the peptides and other agents described herein, including implants (such as implantable pumps) and catheter systems, both of which are known to those skilled in the art. General subcutaneous or intramuscular injections can also be used to release the peptides and/or other agents described herein within a limited time period. Depot injections are generally based on solids or oils, and generally contain at least one of the formulation components described herein. Those who are familiar with this technique are familiar with the possible formulations and usage of depot injections. In some embodiments, the usage of Nano Precision Medical's storage delivery technology (Nano Precision Medical; Emeryville CA) is covered. This technology utilizes titanium dioxide nanotube membranes that produce a zero-order release rate of macromolecules (protein and peptide therapeutics). The biocompatible film is housed in a small subcutaneous implant and provides long-term (eg, up to one year) delivery of therapeutic macromolecules at a constant rate.

The pharmaceutical composition can be formulated according to its intended route of administration. Therefore, the pharmaceutical composition includes excipients suitable for administration by routes including: parenteral (e.g., subcutaneous (sc), intravenous, intramuscular, or intraperitoneal), intradermal, or oral (e.g. ingestion) , Inhalation, intracavity, intracranial and percutaneous (local). This article describes other exemplary investment approaches.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension. This suspension can be formulated using suitable dispersing or wetting agents and suspending agents disclosed herein or known to those skilled in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example in the form of a solution in 1,3-butanediol. Acceptable diluents, solvents and dispersion media that can be used include water, Ringer's solution, isotonic sodium chloride solution, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS) , Ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof. In addition, sterile fixed oils are conventionally used as solvents or suspension media. For this purpose, any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. Prolonged absorption of certain injectable formulations can be achieved by including agents that delay absorption, such as aluminum monostearate or gelatin.

In one embodiment, the pharmaceutical composition provided herein can be administered parenterally by injection, infusion or implantation for local or systemic administration. As used herein, parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.

In one embodiment, the pharmaceutical composition provided herein can be formulated in any form suitable for parenteral administration with any dosage form, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and A solid form suitable for solution or suspension in liquid before injection. Such dosage forms can be prepared according to conventional methods known to those skilled in medical science and technology (see, for example, Remington, The Science and Practice of Pharmacy , see above).

In one embodiment, the pharmaceutical composition intended for parenteral administration may include one or more pharmaceutically acceptable carriers and excipients, including but not limited to aqueous vehicles, water-miscible vehicles , Non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting agents or emulsifiers, Complexing agent, clamping agent or chelating agent, cryoprotectant, freeze-dried protective agent, thickener, pH regulator and inert gas.

In one embodiment, suitable aqueous vehicles include (but are not limited to) water, saline, physiological saline or phosphate buffered saline (PBS), sodium chloride injection, Ringer's injection, isotonic dextrose injection, Sterile water injection, dextrose and lactated Ringer's injection. Non-aqueous vehicles include (but are not limited to) non-volatile oils of plant origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil and Coconut oil medium chain triglycerides, and palm seed oil. Water miscible vehicles include (but are not limited to) ethanol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N -methyl -2-pyrrolidone, N,N -dimethylacetamide and dimethylsulfoxide.

In one embodiment, suitable antimicrobial agents or preservatives include (but are not limited to) phenol, cresol, amalgam, benzyl alcohol, chlorobutanol, methyl and propyl paraben, thimerosal , Benzalkonium chloride (such as benzethonium chloride), methyl paraben and propyl paraben, and sorbic acid. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffers include, but are not limited to, phosphate and citrate. Suitable antioxidants are those as described herein, including bisulfite and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine hydrochloride. Suitable suspending agents and dispersing agents are those described herein, including sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, and polyvinylpyrrolidone. Suitable emulsifiers include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable chelating agents or chelating agents include but are not limited to EDTA. Suitable pH adjusting agents include (but are not limited to) sodium hydroxide, hydrochloric acid, citric acid and lactic acid. Suitable complexing agents include (but are not limited to) cyclodextrins, including α-cyclodextrin, β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin and sulfo Butyl ether 7-β-cyclodextrin (CAPTISOL ^® , CyDex, Lenexa, KS).

In one embodiment, the pharmaceutical compositions provided herein can be formulated for single dose or multiple dose administration. The single-dose formulations are enclosed in ampoules, vials or syringes. Multiple-dose parenteral formulations may contain antimicrobial agents at bacteriostatic or fungal inhibitory concentrations. As known and practiced in the art, all parenteral formulations must be sterile.

In one embodiment, the pharmaceutical composition is provided as a ready-to-use sterile solution. In another embodiment, the pharmaceutical composition is provided in the form of a sterile dry soluble product to be reconstituted with a vehicle before use, including a lyophilized powder and subcutaneous lozenge. In yet another embodiment, the pharmaceutical composition is provided in the form of a ready-to-use sterile suspension. In yet another embodiment, the pharmaceutical composition is provided in the form of a sterile dry insoluble product to be reconstituted with a vehicle before use. In yet another embodiment, the pharmaceutical composition is provided in the form of a ready-to-use sterile emulsion.

In one embodiment, the pharmaceutical composition provided herein can be formulated into an immediate release dosage form or a modified release dosage form, including delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

In one embodiment, the pharmaceutical composition can be formulated as a suspension, solid, semi-solid, or thixotropic liquid for administration in the form of implant storage. In one embodiment, the pharmaceutical composition provided herein is dispersed in a solid internal matrix that is surrounded by an outer polymeric film that is insoluble in body fluids but allows the active ingredients in the pharmaceutical composition to diffuse through.

In one embodiment, suitable internal substrates include polymethyl methacrylate, polybutyl methacrylate, plasticized or non-plasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate , Natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymer, polysilicone rubber, polydimethylsiloxane, polysiloxane carbonate copolymer, Hydrophilic polymers (such as hydrogels of acrylic acid and methacrylic acid esters), collagen, cross-linked polyvinyl alcohol and cross-linked partially hydrolyzed polyvinyl acetate.

In one embodiment, suitable outer polymeric films include polyethylene, polypropylene, ethylene/propylene copolymer, ethylene/ethyl acrylate copolymer, ethylene/vinyl acetate copolymer, polysiloxane rubber, polydimethylsiloxane Oxyane, chloroprene rubber, chlorinated polyethylene, polyvinyl chloride, vinyl chloride-vinyl acetate copolymer, vinyl chloride-vinylidene chloride copolymer, vinyl chloride-ethylene copolymer and vinyl chloride-propylene copolymer, ion Polymer polyethylene terephthalate, butyl rubber epichlorohydrin rubber, ethylene/vinyl alcohol copolymer, ethylene/ethyleneoxyethanol copolymer and ethylene/vinyl acetate/vinyl alcohol terpolymer.

The aqueous suspension contains the active material blended with excipients suitable for the manufacture of the active material. Such excipients are suspending agents, such as sodium carboxymethyl cellulose, methyl cellulose, hydroxy-propyl methyl cellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersing agent Or the wetting agent may be naturally occurring phospholipids, such as lecithin, or condensation products of alkylene oxide and fatty acids (such as polyoxyethylene stearate), or condensation products of ethylene oxide and long-chain aliphatic alcohols (such as seventeen Ethyleneoxyhexadecanol), or condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol (such as polyoxyethylene sorbitol monooleate), or ethylene oxide and condensation products derived from fatty acids and hexitol Condensation products of partial esters of sugar alcohol anhydrides (for example, polyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives.

Oily suspensions can be formulated by suspending the active ingredients in vegetable oils (for example, peanut oil, olive oil, sesame oil, or coconut oil) or mineral oils (such as liquid paraffin). Oily suspensions may contain thickeners such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents (such as those described above) and flavoring agents can be added to provide a palatable oral preparation.

Dispersible powders and granules suitable for preparing aqueous suspensions by adding water provide active ingredients blended with dispersing or wetting agents, suspending agents and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified herein.

The pharmaceutical compositions provided herein may also be in the form of oil-in-water emulsions. The oil phase can be vegetable oil, such as olive oil or peanut oil; or mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifiers can be naturally occurring gums, such as gum arabic or tragacanth; naturally occurring phospholipids, such as soybeans, lecithin, and esters or partial esters derived from fatty acids; and hexitol anhydrides, such as sorbitan Monooleate; and condensation products of partial esters and ethylene oxide, such as polyoxyethylene sorbitan monooleate.

Pharmaceutical compositions may also include excipients to protect the composition from rapid degradation or discharge from the body, such as controlled release formulations, including implants, liposomes, hydrogels, prodrugs, and microencapsulated delivery systems. For example, time delay materials may be used, such as glyceryl monostearate alone or glyceryl stearate or a combination with wax. Prolonged absorption of injectable pharmaceutical compositions can be achieved by including agents that delay absorption, such as aluminum monostearate or gelatin. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.

The pharmaceutical composition provided herein can be stored at -80°C, 4°C, 25°C, or 37°C.

The lyophilized composition can be prepared by freeze-drying the liquid pharmaceutical composition provided herein. In a specific embodiment, the pharmaceutical composition provided herein is a freeze-dried pharmaceutical composition. In some embodiments, the pharmaceutical formulation is a lyophilized powder, which can be reconstituted for administration in the form of solutions, emulsions, and other mixtures. It can also be reconstituted and formulated into a solid or gel form.

In some embodiments, the preparation of the lyophilized formulations provided herein involves batching the bulk solution for lyophilization and aseptic filtration, filling the vials, and freezing the vials in a freeze dryer box , Then freeze-dried, stoppered and capped.

The lyophilizer can be used to prepare lyophilized formulations. For example, the VirTis genetic model EL point unit can be used. The unit incorporates a box with three working shelves (approximately 0.4 square meters of total usable shelf area), external condenser and mechanical vacuum pump system. The cascaded mechanical refrigeration allows the shelf to cool to -70°C or lower, and allows the external condenser to cool to -90°C or lower. Automatically control shelf temperature and box pressure to +/- 0.5°C and +/- 2 microns (mtorr) respectively. The unit is equipped with a vacuum capacitance pressure gauge, Pirani vacuum gauge, pressure converter (measurement range: 0 to 1 atmosphere) and a relative humidity sensor.

The lyophilized powder can be prepared by dissolving the antibody-drug conjugate provided herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial will contain a single dose or multiple doses of antibody drug conjugate. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature.

The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable excipients. This amount can be determined empirically and adjusted according to specific needs.

An exemplary restoration procedure is described as follows: (1) Fit an 18 or 20 gauge needle to a 5 mL or 3 mL syringe and fill the syringe with water for injection (WFI) grade water; (2) Use the syringe scale to measure the appropriate amount of WFI to ensure The syringe does not contain air bubbles; (3) Insert the needle through the rubber stopper; (4) Distribute the entire contents of the syringe down the bottle wall into the container, remove the syringe and needle and place them in the sharps container; (5) Continue to vortex the vial to carefully dissolve the entire vial contents until complete recovery (for example, about 20-40 seconds), and minimize agitation of excessive protein solution that can cause foaming. 5.3 Anti-191P4D12 antibody drug conjugate

The pharmaceutical compositions, formulations and dosage forms provided herein include anti-191P4D12 antibody drug conjugates. The anti-191P4D12 antibody drug conjugate provided herein comprises a 191P4D12-binding antibody or antigen-binding fragment thereof bound to one or more cytotoxic agent (or drug unit) units. The cytotoxic agent (or drug unit) can be covalently attached directly or via a linking unit (LU).

In some embodiments, the antibody-drug conjugate compound has the following formula: L-(LU-D) _p (I) or a pharmaceutically acceptable salt or solvate thereof, wherein: L is an antibody unit, for example, as described below The anti-191P4D12 antibody or its antigen-binding fragment provided in section 5.3.1 of the article, and (LU-D) is the linking unit-drug unit part, where: LU- is the linking unit, and D is the cell growth inhibitory target Or cytotoxic active drug unit; and p is an integer from 1 to 20.

In some embodiments, p is in the range of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is about 1. In other embodiments, p is about 2. In other embodiments, p is about 3. In other embodiments, p is about 4. In other embodiments, p is about 5. In other embodiments, p is about 6. In other embodiments, p is about 7. In other embodiments, p is about 8. In other embodiments, p is about 9. In other embodiments, p is about 10.

In some embodiments, the antibody drug conjugate compound has the following formula: L-(A _a -W _w -Y _y -D) _p (II) or a pharmaceutically acceptable salt or solvate thereof, wherein: L It is an antibody unit, such as the anti-191P4D12 antibody or antigen-binding fragment thereof as provided in Section 5.3.1 below; and -A _a -W _w -Y _y -is the linking unit (LU), where: -A- is the extension Unit, a is 0 or 1, each -W- is independently an amino acid unit, w is an integer in the range of 0 to 12, -Y- is a self-decomposing spacer unit, and y is 0, 1 or 2; D is a drug unit with cell growth inhibitory or cytotoxic activity against target cells; and p is an integer from 1 to 20.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, p is in the range of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is in the range of 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5, or 6. In some embodiments, p is 2 or 4. In some embodiments, when w is not zero, y is 1 or 2. In some embodiments, when w is 1-12, y is 1 or 2. In some embodiments, w is 2-12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0.

For a composition containing multiple antibodies or antigen-binding fragments thereof, the drug load is represented by p (average number of drug molecules/antibody unit). The drug load can be in the range of 1 to 20 drugs (D)/antibody. The average number of drugs/antibodies in the binding reaction preparation can be characterized by conventional means such as mass spectrometry, ELISA analysis and HPLC. The quantitative distribution of antibody-drug conjugates in terms of p can also be determined. In some cases, the separation, purification and characterization of homogeneous antibody-drug conjugates where p is a certain value of antibody-drug conjugates loaded with other drugs can be achieved by means such as reverse phase HPLC or electrophoresis. In an exemplary embodiment, p is 2-8. 5.3.1 Anti- 191P4D12 antibody or antigen-binding fragment

In one embodiment, the antibody or antigen-binding fragment thereof that binds to a 191P4D12-related protein is an antibody or antigen-binding fragment that specifically binds to the 191P4D12 protein comprising the amino acid sequence of SEQ ID NO: 2 (see Figure 5A). The corresponding cDNA encoding the 191P4D12 protein has the sequence of SEQ ID NO: 1 (see Figure 5A).

Antibodies that specifically bind to the 191P4D12 protein comprising the amino acid sequence of SEQ ID NO: 2 include antibodies that can bind to other 191P4D12 related proteins. For example, an antibody that binds to the 191P4D12 protein comprising the amino acid sequence of SEQ ID NO: 2 can bind to a 191P4D12 related protein (such as a 191P4D12 variant) and homologs or analogs thereof.

In some embodiments, the anti-191P4D12 antibodies provided herein are monoclonal antibodies.

In some embodiments, the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 (the cDNA sequence of SEQ ID NO: 3); and/or a light chain comprising SEQ ID NO: 6 (SEQ ID NO: 6) The amino acid sequence of ID NO: 5 (cDNA sequence) is shown in Figure 5B.

SEQ ID NO:8

In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising a complementarity determining region containing the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO:7 (CDR); and the light chain variable region, which includes a CDR containing the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO:8. SEQ ID NO: 7 and SEQ ID NO: 8 are shown in Figure 5C and are listed below: SEQ ID NO: 7

SEQ ID NO: 8

SEQ ID NO:23

In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising SEQ ID NO: 22 (which is the 20th amino acid (glutamate) in SEQ ID NO: 7) to The amino acid sequence of the heavy chain variable region set forth in the 136th amino acid (amino acid sequence within the range of serine); and the light chain variable region, which includes SEQ ID NO: 23 (its For the light chain variable region set forth in SEQ ID NO: 8 from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) in the amino acid sequence) Amino acid sequence. In other embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region consisting of SEQ ID NO: 22 (which is the 20th amino acid (glutamine) in SEQ ID NO: 7) The amino acid sequence of the heavy chain variable region described in the amino acid sequence within the range of the 136th amino acid (serine)); and the light chain variable region, which consists of SEQ ID NO: 23 (It is the amino acid sequence within the range of the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) of SEQ ID NO: 8) as described in the variable light chain The amino acid sequence of the region is composed. SEQ ID NO: 22 and SEQ ID NO: 23 are listed as follows: SEQ ID NO: 22

SEQ ID NO: 23

The CDR sequence can be determined according to a well-known numbering system. As described above, CDR regions are well known to those skilled in the art and have been defined by a well-known numbering system. For example, the Kabat complementarity determining region (CDR) is based on sequence variability and is most commonly used (see, for example, Kabat et al., supra). Chothia relates to the position of structural loops (see, for example, Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). When using the Kabat numbering convention for numbering, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion in H35A and H35B; if neither exists If 35A and 35B are not present, the end of the ring is at 32; if only 35A is present, the end of the ring is at 33; if both 35A and 35B are present, the end of the ring is at 34). The AbM hypervariable region represents the balance between the Kabat CDR and the Chothia structural loop, and is used by Oxford Molecular's AbM antibody modeling software (see, for example, Antibody Engineering Vol. 2 (Kontermann and Dübel eds, 2nd edition, 2010)). The "contact" hypervariable zone is based on the analysis of available composite crystal structures. Another universal numbering system that has been developed and widely adopted is ImmunoGeneTics (IMGT) Information ^System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). IMGT is an integrated information system dedicated to immunoglobulin (IG), T cell receptor (TCR) and major histocompatibility complex (MHC) of humans and other vertebrates. Herein, CDRs are mentioned with regard to the amino acid sequence and the position in the light chain or heavy chain. Since the "position" of the CDR in the immunoglobulin variable domain structure is conserved between species and exists in a structure called a loop, it is easy to identify the CDR using a numbering system that aligns the variable domain sequence according to structural features And framework residues. This information can be used to transplant and replace CDR residues from immunoglobulins from a species into the acceptor framework typically derived from human antibodies. Honegger and Plückthun, 2001, J. Mol. Biol. 309: 657-70 have developed another numbering system (AHon). The correspondence between numbering systems (including, for example, Kabat numbering and IMGT unique numbering systems) is well known to those skilled in the art (see, for example, Kabat, see above; Chothia and Lesk, see above; Martin, see above; Lefranc Et al., see above). The residues from each of these hypervariable regions or CDRs are indicated in Table 30 above.

In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence containing the CDR of the heavy chain variable region set forth in SEQ ID NO: 7 according to Kabat numbering The complementarity determining region (CDR); and the light chain variable region, which comprises a CDR containing the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 8 numbered according to Kabat.

In some embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence containing the CDR of the heavy chain variable region set forth in SEQ ID NO: 7 numbered according to AbM The complementarity determining region (CDR); and the light chain variable region, which comprises a CDR containing the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 8 numbered according to AbM.

In other embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence containing the CDR of the heavy chain variable region set forth in SEQ ID NO: 7 according to Chothia numbering The complementarity determining region (CDR); and the light chain variable region comprising a CDR containing the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 8 numbered according to Chothia.

In other embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence containing the CDR of the heavy chain variable region set forth in SEQ ID NO: 7 according to Contact numbering The complementarity determining region (CDR); and the light chain variable region, which includes a CDR containing the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 8 numbered according to Contact.

In still other embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid containing the CDR of the heavy chain variable region set forth in SEQ ID NO: 7 numbered according to IMGT The complementarity determining region (CDR) of the sequence; and the light chain variable region, which comprises a CDR containing the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 8 numbered according to IMGT.

As described above, CDR sequences according to different numbering systems can be easily determined, for example, using online tools, such as the tools provided by Antigen Receptor Numbering and Receptor Classification (ANARCI). For example, as determined by ANARCI, the heavy chain CDR sequence in SEQ ID NO: 7 and the light chain CDR sequence in SEQ ID NO: 8 according to Kabat numbering are listed in Table 31 below. Table 31 SEQ ID NO: VH of 7 SEQ ID NO: VL of 8 CDR1 SYNMN (SEQ ID NO: 9) RASQGISGWLA (SEQ ID NO: 12) CDR2 YISSSSSTIYYADSVKG (SEQ ID NO: 10) AASTLQS (SEQ ID NO: 13) CDR3 AYYYGMDV (SEQ ID NO: 11) QQANSFPPT (SEQ ID NO: 14)

For another example, as determined by ANARCI, the heavy chain CDR sequence in SEQ ID NO: 22 and the light chain CDR sequence in SEQ ID NO: 23 numbered according to IMGT are listed in Table 32 below. Table 32 SEQ ID NO: VH of 7 SEQ ID NO: VL of 8 CDR1 GFTFSSYN (SEQ ID NO: 16) QGISGW (SEQ ID NO: 19) CDR2 ISSSSSTI (SEQ ID NO: 17) AAS (SEQ ID NO: 20) CDR3 ARAYYYGMDV (SEQ ID NO: 18) QQANSFPPT (SEQ ID NO: 21)

In some embodiments, the antibody or antigen-binding fragment thereof comprises: CDR H1, which comprises the amino acid sequence of SEQ ID NO: 9; CDR H2, which comprises the amino acid sequence of SEQ ID NO: 10; CDR H3, which Comprises the amino acid sequence of SEQ ID NO: 11; CDR L1, which comprises the amino acid sequence of SEQ ID NO: 12; CDR L2, which comprises the amino acid sequence of SEQ ID NO: 13; and CDR L3, which comprises The amino acid sequence of SEQ ID NO: 14.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the 20th amino acid (glutamate) to the 136th amino acid (seramine) of SEQ ID NO: 7 Acid) within the range of amino acid sequence; and the light chain variable region, which is included in SEQ ID NO: 8 from the 23rd amino acid (aspartic acid) to the 130th amino acid (arginine) Amino acid sequence within the range.

In some embodiments, the antibody comprises: a heavy chain comprising an amino acid in the range of the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO: 7 Sequence; and light chain, which includes the amino acid sequence in the range of the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO: 8.

In some embodiments, modifications to the amino acid sequence of the antibodies described herein are covered. For example, it may be necessary to optimize the binding affinity and/or other biological properties of the antibody, including (but not limited to) specificity, thermal stability, performance, effector function, glycosylation, reduced immunogenicity or solubilization Sex. Therefore, in addition to the antibodies described herein, it is expected that antibody variants can be prepared. For example, antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA and/or by synthesizing the desired antibody or polypeptide. Those familiar with the art of amino acid changes can change the post-translational process of antibodies, such as changing the number or location of glycosylation sites or changing membrane anchoring characteristics.

In some embodiments, the antibodies provided herein are chemically modified, for example, by covalently linking any type of molecule to the antibody. Antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amination, derivatization by known protecting groups/capping groups, protein Cleavage, link to cell ligands or other proteins, etc. and chemically modified antibodies. Any of a variety of chemical modifications can be performed by known techniques, including (but not limited to) specific chemical lysis, acetylation, formylation, and metabolic synthesis of tunicamycin. In addition, antibodies may contain one or more non-classical amino acids.

The variation may be a substitution, deletion, or insertion of one or more codons of a single-domain antibody or polypeptide encoding a single-domain antibody or polypeptide that results in an amino acid sequence change compared with the original antibody or polypeptide. Amino acid substitution may be the result of one amino acid being replaced by another amino acid containing similar structure and/or chemical characteristics, such as leucine replacement by serine, such as conservative amino acid replacement. Standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the molecules provided herein, including, for example, site-directed mutagenesis that causes amino acid substitutions and PCR-mediated mutagenesis. The insertion or deletion can be in the range of about 1 to 5 amino acids depending on the situation. In certain embodiments, relative to the original molecule, the substitution, deletion or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, and less than 10 amines. Base acid substitution, less than 5 amino acid substitution, less than 4 amino acid substitution, less than 3 amino acid substitution, or less than 2 amino acid substitution. In a specific embodiment, the substitutions are conservative amino acid substitutions at one or more predicted non-essential amino acid residues. Allowable variation can be determined by systematically generating amino acid insertions, deletions or substitutions in the sequence and testing the activity exhibited by the parent antibody of the resulting variant.

Amino acid sequence insertions include amino-terminal and/or carboxy-terminal fusions ranging from one residue to a polypeptide containing multiple residues, and insertions within the sequence of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionine residues.

Antibodies produced by conservative amino acid substitutions are included in the present invention. In conservative amino acid substitutions, the amino acid residue is replaced with an amino acid residue containing a side chain with similar charge. As described above, the family of amino acid residues containing side chains with similar charges has been defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamine), and non-electrode side chains (e.g., glycine acid). , Aspartame, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine) , Proline, amphetamine, methionine, tryptophan), beta branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, amphetamine) Acid, tryptophan, histidine) of the amino acid. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutations, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed, and the activity of the protein can be measured. Conservative (for example, in amino acid groups with similar characteristics and/or side chains) substitutions can be made to maintain or not change significantly characteristic.

Amino acids can be grouped according to the similarity of their side chain properties (see, for example, Lehninger, Biochemistry 73-75 (2nd edition 1975)): (1) Non-polar: Ala (A), Val (V), Leu (L) , Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) Without electrical polarity: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) Acidic: Asp (D), Glu (E); and (4) Basic: Lys (K), Arg (R) , His (H). Alternatively, naturally-occurring residues can be grouped based on common side chain characteristics: (1) Hydrophobicity: ortholeucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatics: Trp, Tyr, Phe.

For example, any cysteine residues that are not involved in maintaining the proper conformation of the antibody can also be substituted with, for example, another amino acid (such as alanine or serine) to improve the oxidative stability of the molecule and prevent abnormal interactions. United.

Methods known in the art can be used to generate mutations, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis can be performed on the selected DNA (see, for example, Carter, 1986, Biochem J. 237:1-7; and Zoller et al., 1982, Nucl. Acids Res. 10:6487-500), cassette mutagenesis (See, for example, Wells et al., 1985, Gene 34:315-23) or other known techniques to generate anti-anti-MSLN antibody variant DNA.

Covalent modification of antibodies is included in the scope of the present invention. Covalent modification involves reacting the target amino acid residue of the antibody with an organic derivatizing agent that can react with the selected side chain or N-terminal or C-terminal residue of the antibody. Other modifications include deamidation of glutamine and aspartame residues to corresponding glutamine and aspartame residues; hydroxylation of proline and lysine; seramine Phosphorylation of hydroxyl groups or threonyl residues; methylation of α-amino groups of lysine, arginine and histidine side chains (see, for example, Creighton, Proteins: Structure and Molecular Properties 79-86 (1983)); acetylation of N-terminal amine; and amination of any C-terminal carboxyl group.

Other types of covalent modifications of antibodies included within the scope of the present invention include changing the native glycosylation pattern of antibodies or polypeptides (see, for example, Beck et al., 2008, Curr. Pharm. Biotechnol. 9:482-501; and Walsh, 2010, Drug Discov. Today 15:773-80), and combine the antibody with a variety of non-proteinaceous polymers (such as polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene) in the manner described in the following documents. One connection: US Patent No. 4,640,835, No. 4,496,689, No. 4,301,144, No. 4,670,417, No. 4,791,192 or No. 4,179,337.

In some embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain having greater than 70% homology with the heavy chain as set forth in SEQ ID NO:7. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain having greater than 75% homology with the heavy chain as set forth in SEQ ID NO:7. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain having greater than 80% homology with the heavy chain as set forth in SEQ ID NO:7. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain having greater than 85% homology with the heavy chain as set forth in SEQ ID NO:7. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain having greater than 90% homology with the heavy chain as set forth in SEQ ID NO:7. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain having greater than 95% homology with the heavy chain as set forth in SEQ ID NO:7.

In some embodiments, the antibody or antigen-binding fragment provided herein comprises a light chain having greater than 70% homology with the light chain as set forth in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a light chain having greater than 75% homology with the light chain as set forth in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a light chain having greater than 80% homology with the light chain as set forth in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a light chain having greater than 85% homology to the light chain as set forth in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a light chain having greater than 90% homology with the light chain as set forth in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a light chain having greater than 95% homology with the light chain as set forth in SEQ ID NO:8.

In some embodiments, the anti-191P4D12 antibody provided herein includes: an antibody called Ha22-2(2,4)6.1 (by depositing with the American Type Conservation Center (ATCC) deposit number: PTA-11267 fusion tumor Generated) heavy chain and light chain CDR regions, or heavy chain and light chain CDRs comprising amino acid sequences homologous to the amino acid sequences of the heavy chain and light chain CDR regions of Ha22-2(2,4)6.1 Area, and the antibody in it retains the required function of the anti-191P4D12 antibody called Ha22-2(2,4)6.1 (produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267) characteristic.

In some embodiments, the antibodies or antigen-binding fragments thereof provided herein comprise a humanized heavy chain variable region and a humanized light chain variable region, wherein: (a) The heavy chain variable region contains the amino acid sequence containing the CDR of the heavy chain variable region described in the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 CDR; (b) The light chain variable region contains the amino acid sequence containing the CDR of the light chain variable region described in the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 CDR.

In some embodiments, the anti-191P4D12 antibody provided herein includes: an antibody called Ha22-2(2,4)6.1 (by depositing with the American Type Conservation Center (ATCC) deposit number: PTA-11267 fusion tumor Generated) heavy chain and light chain variable regions (see Figure 5), or include amino acid sequences homologous to the amino acid sequences of the heavy chain and light chain variable regions of Ha22-2(2,4)6.1 The variable regions of the heavy chain and light chain of and wherein the antibody retains the desired functional properties of the anti-191P4D12 antibody provided herein. Any subclass of the constant region can be selected as the constant region of the antibody of the present invention. In one embodiment, the human IgG1 constant region can be used as the heavy chain constant region and the human Igκ constant region can be used as the light chain constant region.

In some embodiments, the anti-191P4D12 antibody provided herein includes: an antibody called Ha22-2(2,4)6.1 (by depositing with the American Type Conservation Center (ATCC) deposit number: PTA-11267 fusion tumor Produced) heavy chain and light chain (see Figure 5), or heavy chain and light chain containing amino acid sequences homologous to the amino acid sequences of the heavy and light chains of Ha22-2(2,4)6.1 , And wherein the antibody retains the desired functional properties of the anti-191P4D12 antibody provided herein.

In some embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region and a light chain variable region, wherein: (a) The heavy chain variable region contains at least 80% homology with the amino acid sequence of the heavy chain variable region of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 The amino acid sequence of; and (b) The light chain variable region contains at least 80% homology with the amino acid sequence of the light chain variable region of the antibody generated by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 The amino acid sequence.

In some embodiments, the heavy chain variable region comprises at least 85 amino acid sequences of the heavy chain variable region of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 % Homologous amino acid sequence. In other embodiments, the heavy chain variable region comprises at least 90 amino acid sequences of the heavy chain variable region of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 % Homologous amino acid sequence. In still other embodiments, the heavy chain variable region comprises at least the amino acid sequence of the heavy chain variable region of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 95% homologous amino acid sequence. In other embodiments, the heavy chain variable region can be combined with 85% of the amino acid sequence of the heavy chain variable region of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

In some embodiments, the light chain variable region contains at least 85 amino acid sequences of the light chain variable region of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 % Homologous amino acid sequence. In other embodiments, the light chain variable region comprises at least 90 amino acid sequences of the light chain variable region of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 % Homologous amino acid sequence. In still other embodiments, the light chain variable region comprises at least the amino acid sequence of the light chain variable region of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 95% homologous amino acid sequence. In other embodiments, the light chain variable region can be combined with 85% of the amino acid sequence of the light chain variable region of the antibody generated by the fusion tumor deposited under the American Culture Collection (ATCC) under the deposit number: PTA-11267 , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

In other embodiments, the antibody or antigen-binding fragment thereof provided herein comprises a heavy chain and a light chain, wherein: (a) The heavy chain contains an amino acid sequence that is at least 80% homologous to the heavy chain amino acid sequence of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267; and (b) The light chain contains an amino acid sequence that is at least 80% homologous to the light chain amino acid sequence of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267.

In some embodiments, the heavy chain contains an amino group that is at least 85% homologous to the heavy chain amino acid sequence of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 Acid sequence. In other embodiments, the heavy chain contains an amino group that is at least 90% homologous to the heavy chain amino acid sequence of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 Acid sequence. In still other embodiments, the heavy chain comprises an amine that is at least 95% homologous to the heavy chain amino acid sequence of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 Base acid sequence. In other embodiments, the heavy chain can be combined with 85%, 86%, 87% of the heavy chain amino acid sequence of the antibody produced by the fusion tumor deposited under the ATCC deposit number: PTA-11267 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

In some embodiments, the light chain contains an amino group that is at least 85% homologous to the amino acid sequence of the light chain of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 Acid sequence. In other embodiments, the light chain contains an amino group that is at least 90% homologous to the amino acid sequence of the light chain of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 Acid sequence. In still other embodiments, the light chain comprises an amine that is at least 95% homologous to the amino acid sequence of the light chain of the antibody produced by the fusion tumor deposited with the American Culture Collection (ATCC) under the deposit number: PTA-11267 Base acid sequence. In other embodiments, the light chain can be combined with 85%, 86%, 87% of the light chain amino acid sequence of the antibody produced by the fusion tumor deposited under the American Type Conservation Center (ATCC) deposit number: PTA-11267 , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

The engineered antibodies provided herein include those in which the framework residues in VH and/or VL have been modified (for example, to improve the properties of the antibody). Generally, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one method is to "backmutate" one or more framework residues to corresponding germline sequences. More specifically, antibodies that have undergone somatic mutations may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence with the germline sequence from which the antibody is derived. In order to restore the framework region sequence to its germline configuration, somatic mutations can be "backmutated" into germline sequences by, for example, site-directed mutagenesis or PCR-mediated mutagenesis (for example, leucine "back mutation" into armour Thiamine). Such "backmutated" antibodies are also intended to be covered by the present invention.

Another type of framework modification involves mutating one or more residues in the framework region or even one or more CDR regions to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This method is also called "deimmunization" and is described in further detail in US Patent Publication No. 2003/0153043 by Carr et al.

In addition to or alternatively to modifications made in the framework or CDR regions, the antibodies of the present invention can be engineered to include modifications in the Fc region, typically to change one or more of the functional properties of the antibody, such as serum half-life, Complement binding, Fc receptor binding and/or antigen-dependent cytotoxicity. In addition, the anti-191P4D12 antibodies provided herein can be chemically modified (for example, one or more chemical moieties can be linked to the antibody) or modified to change its glycosylation, so as to again change one or more functional properties of the antibody. Each of these embodiments is described in further detail below.

In one embodiment, the hinge region of CH1 is modified so that the number of cysteine residues in the hinge region is changed, for example, increased or decreased. This method is further described in U.S. Patent No. 5,677,425 by Bodmer et al. Changing the number of cysteine residues in the CH1 hinge region can, for example, promote the assembly of the light chain and the heavy chain or increase or decrease the stability of the anti-191P4D12 antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the anti-191P4D12 antibody. More specifically, the introduction of one or more amino acid mutations into the CH2-CH3 domain interface region of the Fc hinge fragment makes the binding of the antibody to Staphylococcus protein A (SpA) relative to the binding of the native Fc hinge domain SpA Weaken. This method is described in further detail in US Patent No. 6,165,745 by Ward et al.

In another embodiment, the anti-191P4D12 antibody is modified to increase its biological half-life. Many methods are possible. For example, mutations can be introduced as described in Ward's US Patent No. 6,277,375. Alternatively, in order to increase the biological half-life, the antibody can be changed in the CH1 or CL region to contain the rescue receptor binding epitope of the two loops of the CH2 domain of the Fc region of IgG, as described in US Patent No. 1 of Presta et al. No. 5,869,046 and No. 6,121,022.

In still other embodiments, the Fc region is changed by replacing at least one amino acid residue with a different amino acid residue to change the effector function of the antibody. For example, one or more amino acids selected from specific amino acid residues can be replaced with different amino acid residues, so that the affinity of the antibody to the effector ligand is changed but the antigen binding ability of the parent antibody is retained. The effector ligand with altered affinity can be, for example, the Fc receptor or the C1 component of complement. This method is described in further detail in, for example, US Patent Nos. 5,624,821 and 5,648,260 to Winter et al.

The reactivity of anti-191P4D12 antibody and 191P4D12-related protein can be established by a variety of well-known methods, including Western blotting, immunoprecipitation, ELISA and FACS analysis, using (as needed) 191P4D12-related protein, 191P4D12 expressing cells or their extracts . The 191P4D12 antibody or fragments thereof can be labeled with a detectable label or combined with a second molecule. Suitable detectable labels include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelating agents or enzymes. In addition, methods generally known in the art are used to generate bispecific antibodies specific for two or more 191P4D12 epitopes. Homodimeric antibodies can also be produced by cross-linking techniques known in the art (for example, Wolff et al., Cancer Res. 53: 2560-2565).

In yet another specific embodiment, the anti-191P4D12 antibody provided herein is an antibody comprising the heavy chain and light chain of the antibody designated Ha22-2(2,4)6.1. The heavy chain of Ha22-2(2,4)6.1 consists of an amino acid sequence in the range of the 20th residue (E) to the 466th residue (K) of SEQ ID NO: 7; and Ha22-2 (2,4) The light chain of 6.1 consists of an amino acid sequence ranging from the 23rd residue (D) to the 236th residue (C) of SEQ ID NO:8.

In August 18, 2010, will produce called Ha22-2 (2,4) hybridoma antibody 6.1 of sending (via Federal Express) to PO Box 1549, Manassas, VA 20108 The American Type Culture Collection (ATCC) , And assign the deposit number PTA-11267 . 5.3.2 Cytotoxic agents ( drug unit )

In some embodiments, the ADC comprises an antibody or antigen-binding fragment thereof that binds to donotin or donotin peptide analogs and derivative auristatin (US Patent Nos. 5,635,483; No. 5,780,588). Apnea toxin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear division and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anticancer ( US 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dopetoxin or auristatin drug unit can be connected to the antibody via the N (amino) end or C (carboxy) end of the peptide drug unit (WO 02/088172).

Exemplary auristatin examples include drug units DE and DF linked to the N-terminus of monomethyl auristatin. These drug units were disclosed in Senter et al., "Proceedings of the American Association for Cancer" provided on March 28, 2004. Research", Volume 45, Abstract No. 623, and described in US Patent Publication No. 2005/0238649, the disclosure of which is expressly incorporated by reference in its entirety.

In some embodiments, auristatin is MMAE (where the wavy line indicates the covalent attachment to the linker of the antibody drug conjugate).

In some embodiments, an exemplary embodiment comprising MMAE and linker components (further described herein) has the following structure (where L represents an antibody and p is in the range of 1 to 12):

Generally, peptide-based drug units can be prepared by forming peptide bonds between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method well known in the field of peptide chemistry (see E. Schröder and K. Lübke, "The Peptides", Vol. 1, pp. 76-136, 1965, Academic Press). The Auristatin/Deratoxin drug unit can be prepared according to the method of the following documents: US 5635483; US 5780588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti -Cancer Drug Design 13:243-277; Pettit, GR et al., Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003 ) Nat Biotechnol 21(7):778-784. 5.3.3 Linker

Generally, an antibody drug conjugate includes a linker unit between the drug unit (e.g., MMAE) and the antibody unit (e.g., anti-191P4D12 antibody or antigen-binding fragment thereof). In some embodiments, the linker is cleavable under intracellular conditions such that the linker is cleaved in the intracellular environment to release the drug unit from the antibody. In yet other embodiments, the linker unit is not cleavable and releases the drug, for example, by antibody degradation.

In some embodiments, the linker is cleavable by a lytic agent present in the intracellular environment (eg, lysosome or endosome or caveolae). The linker can be, for example, a peptidyl linker that can be cleaved by intracellular peptidases or proteases (including but not limited to lysosomal or endosomal proteases). In some embodiments, the length of the peptidyl linker is at least two amino acids or at least three amino acids. Lysis agents may include cathepsin B, cathepsin D, and plasmin, which are known to hydrolyze dipeptide drug derivatives and cause the release of active drugs inside target cells (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The most typical peptidyl linker is a peptidyl linker that can be cleaved by enzymes present in 191P4D12 expressive cells. For example, a peptidyl linker (such as Phe-Leu or Gly-Phe-Leu-Gly linker (SEQ ID NO: 15)) that can be cleaved by the thiol-dependent protease cathepsin B can be used. Highly expressed in cancer tissues. Other examples of such linkers are described in, for example, US Patent No. 6,214,345, which is incorporated herein by reference in its entirety and for all purposes. In a specific embodiment, the peptidyl linker that can be cleaved by intracellular proteases is the Val-Cit linker or the Phe-Lys linker (see, for example, US Patent 6,214,345, which describes the synthesis of cranberries using the Val-Cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is generally less toxic when conjugated and the serum stability of the conjugate is generally higher.

In other embodiments, the cleavable linker is pH sensitive, that is, sensitive to hydrolysis at certain pH values. Generally, pH-sensitive linkers can be hydrolyzed under acidic conditions. For example, acid-labile linkers that can be hydrolyzed in the lysosome (e.g., hydrazone, hemicarbazone, thiosemicarbazone, cis-aconitine, orthoester, acetal, ketal, or the like By). (See, for example, US Patent No. 5,122,368; No. 5,824,805; No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123; Neville et al., 1989, Biol. Chem. 264: 14653-14661.) Class linkers are relatively stable under neutral pH conditions, such as those in blood, but are unstable below pH 5.5 or 5.0 (approximately lysosome pH). In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether linked to a therapeutic agent via a hydrazone bond (see, for example, US Patent No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducing conditions (e.g., disulfide linker). Various disulfide linkers are known in the art, including, for example, N-succinimino-S-acetylthioacetate (SATA), N-succinimino -3-(2-pyridyldisulfide) propionate (SPDP), N-butanediimidate-3-(2-pyridyldisulfide)butyrate (SPDB) and N-butanedi These linkers are formed by amimino-oxycarbonyl-α-methyl-α-(2-pyridyl-disulfide)toluene (SMPT), SPDB and SMPT. (See, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935 number.)

In still other specific embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleiminobenzyl linker (Lau Et al., 1995, Bioorg-Med-Chem. 3(10): 1299-1304) or 3'-N-amide analogs (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305- 12).

In still other embodiments, the linker unit is not cleavable and is degraded by the antibody to release the drug. (See U.S. Publication No. 2005/0238649, which is incorporated herein by reference in its entirety and for all purposes).

Generally, linkers are substantially insensitive to the extracellular environment. As used herein, in the case of linkers, "substantially insensitive to the extracellular environment" means that the antibody-drug conjugate sample is not more than about 20%, usually not more than about 15%, and more usually not more than about 10%. % And even more usually not more than about 5%, not more than about 3%, or not more than about 1% of the linker is cleaved when the antibody drug conjugate is present in the extracellular environment (eg plasma). Whether the linker is substantially insensitive to the extracellular environment can be achieved, for example, by incubating the antibody-drug conjugate compound with plasma for a predetermined period of time (for example, 2, 4, 8, 16, or 24 hours), and then quantifying the presence in the plasma The amount of free drug is determined.

In other non-mutually exclusive embodiments, the linker promotes cell internalization. In certain embodiments, when combined with a therapeutic agent (ie, in the context of the linker-therapeutic agent portion of the antibody-drug conjugate compound as described herein), the linker promotes cellular internalization. In yet other embodiments, the linker promotes cell internalization when combined with both the auristatin compound and the anti-191P4D12 antibody or antigen-binding fragment thereof.

Various exemplary linkers that can be used with the compositions and methods of the present invention are described in WO 2004-010957, U.S. Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S. Publication No. 2006/0024317 (among them Each is incorporated herein by reference in its entirety and for all purposes).

A "linker unit" (LU) is a bifunctional compound that can be used to connect a drug unit and an antibody unit to form an antibody-drug conjugate. In some embodiments, the linker unit has the following formula: -A _a -W _w -Y _y -where: -A- is an extension unit, a is 0 or 1, and each -W- is independently an amino acid unit , W is an integer in the range of 0 to 12, -Y- is a self-decomposing spacer unit, and y is 0, 1, or 2.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1, or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In some embodiments, when w is 1-12, y is 1 or 2. In some embodiments, w is 2-12 and y is 1 or 2. In some embodiments, a is 1 and w and y are 0. 5.3.3.1 Extension subunit

The extension subunit (A) (when present) can combine the antibody unit with the amino acid unit (-W-) (if present), with the spacer unit (-Y-) (if present), or with the drug unit (-D). )connection. Applicable functional groups that can exist naturally or through chemical manipulations on the anti-191P4D12 antibody or its antigen-binding fragment (e.g. Ha22-2(2,4)6.1) include (but are not limited to) sulfhydryl, amine, hydroxyl, The mutagenic hydroxyl and carboxyl groups of carbohydrates. Suitable functional groups are sulfhydryl and amino groups. In one example, the sulfhydryl group can be generated by reducing the intramolecular disulfide bond of the anti-191P4D12 antibody or antigen-binding fragment thereof. In another embodiment, the sulfhydryl group can be obtained by the amine group of the lysine moiety of the anti-191P4D12 antibody or antigen-binding fragment and 2-iminothiolane (Traut's reagent) or Other sulfhydryl generating reagents react to produce. In certain embodiments, the anti-191P4D12 antibody or antigen-binding fragment thereof is a recombinant antibody and is engineered to carry one or more lysine acids. In certain other embodiments, the recombinant anti-191P4D12 antibody is engineered to carry additional sulfhydryl groups, such as additional cysteine.

In one embodiment, the extension subunit forms a bond with the sulfur atom of the antibody unit. The sulfur atom can be derived from the sulfhydryl group of the antibody. The representative extension subunit of this embodiment is depicted in the square brackets of the following formulas IIIa and IIIb, wherein L-, -W-, -Y-, -D, w, and y are as defined above, and R ¹⁷ is selected from -C ₁ -C ₁₀ alkylene-, -C ₁ -C ₁₀ alkenylene-, -C ₁ -C ₁₀ alkynylene-, carbocyclic-, -O-(C ₁ -C ₈ alkylene) -, O-(C ₁ -C ₈ Alkenylene)-, -O-(C ₁ -C ₈ Alkynylene)-, -Arylene-, -C ₁ -C ₁₀ Alkenylene-Arylene -, -C ₂ -C ₁₀ Alkenylene-Arylene, -C ₂ -C ₁₀ Alkynylene-Arylene,-Arylene-C ₁ -C ₁₀ Alkenylene -,-Arylene- C ₂ -C ₁₀ Alkenylene -, -Arylene -C ₂ -C ₁₀ Alkynylene -, -C ₁ -C ₁₀ Alkenylene-(Carbocyclic) -, -C ₂ -C ₁₀ Alkenylene -(Carbocyclic)-, -C ₂ -C ₁₀ Alkynylene-(Carbocyclic)-, -(Carbocyclic)-C ₁ -C ₁₀ Alkylene-, -(Carbocyclic)-C ₂ -C ₁₀ Alkenylene-, -(carbocyclic)-C ₂ -C ₁₀ alkynylene, -heterocyclic-, -C ₁ -C ₁₀ alkylene-(heterocyclic)-, -C ₂ -C ₁₀ alkenylene -(Heterocycle)-, -C ₂ -C ₁₀ Alkynylene-(Heterocycle)-, -(Heterocycle)-C ₁ -C ₁₀ Alkylene-, -(Heterocycle)-C ₂ -C ₁₀ Alkenylene-, -(heterocyclic)-C ₁ -C ₁₀ alkynylene-, -(CH ₂ CH ₂ O) _r -or -(CH ₂ CH ₂ O) _r -CH ₂ -, and r is in An integer in the range of 1-10, where the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic, carbocyclic, heterocyclic and aryl (regardless of Alone or as part of another group) optionally substituted. In some embodiments, the alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, aryl, carbocyclic, carbocyclic, heterocyclic, and arylene groups (whether alone or as Part of the other group) is unsubstituted.

In some embodiments, R ¹⁷ is selected from -C ₁ -C ₁₀ alkylene-, -carbocyclic-, -O-(C ₁ -C ₈ alkylene)-, -arylene-, -C ₁ -C ₁₀ alkylene-arylene-, -arylene-C ₁ -C ₁₀ alkylene-, -C ₁ -C ₁₀ alkylene-(carbocyclic)-, -(carbocyclic)- C ₁ -C ₁₀ alkylene-, -C ₃ -C ₈ heterocyclic-, -C ₁ -C ₁₀ alkylene-(heterocyclic)-, -(heterocyclic)-C ₁ -C ₁₀ alkylene -, -(CH ₂ CH ₂ O) _r -and -(CH ₂ CH ₂ O) _r -CH ₂ -; and r is an integer in the range of 1-10, wherein these alkylene groups are unsubstituted, and The remaining groups are optionally substituted.

It should be understood from all the exemplary embodiments that, even if not explicitly indicated, 1 to 20 drug units can be connected to the antibody unit (p=1-20).

。An illustrative extension subunit is an extension subunit of formula IIIa, wherein R ¹⁷ is -(CH ₂ ) ₅ -:

.

。Another illustrative extension subunit is the extension subunit of formula IIIa, wherein R ¹⁷ is -(CH ₂ CH ₂ O) _r -CH ₂ -; and r is 2:

.

An illustrative extension subunit is an extension subunit of formula IIIa, wherein R ¹⁷ is an aryl- or aryl-C ₁ -C ₁₀ alkylene-: in some embodiments, the aryl group is an unsubstituted benzene base.

。Another illustrative extension subunit is the extension subunit of formula IIIb, wherein R ¹⁷ is -(CH ₂ ) ₅ -:

.

In certain embodiments, the extension subunit is connected to the antibody unit via a disulfide bond between the sulfur atom of the antibody unit and the sulfur atom of the extension subunit. The representative extension subunit of this embodiment is depicted in the square brackets of Formula IV, where R ¹⁷ , L-, -W-, -Y-, -D, w, and y are as defined above.

It should be noted that throughout this application, unless the context indicates otherwise, the S part in the following formula refers to the sulfur atom of the antibody unit.

In the description of certain structures of sulfur-linked ADCs herein, antibodies are denoted as "L". It can also be indicated as "Ab-S". The inclusion of "S" only indicates the sulfur linkage characteristics, and does not indicate that a specific sulfur atom carries multiple linker-drug moieties. The left side parenthesis of the structure can be described using "Ab-S" and can also be placed between Ab and S on the left side of the sulfur atom, which should be the equivalent description of the ADC of the present invention described throughout this text.

In still other embodiments, the extender contains a reactive site that can form a bond with the primary or secondary amine group of the antibody unit. Examples of such reactive sites include, but are not limited to, activated esters such as succinimide, 4-nitrophenyl, pentafluorophenyl, tetrafluorophenyl, acid anhydrides, acid chlorides, sulfonated chlorides , Isocyanates and isothiocyanates. The representative extension subunits of this embodiment are depicted in the square brackets of formulas Va and Vb, where -R ¹⁷ -, L-, -W-, -Y-, -D, w and y are as defined above;

5.3.3.2 胺基酸單元 In some embodiments, the extender contains a reactive site that is reactive with the (-CHO) group of the modified carbohydrate that may be present on the antibody unit. For example, carbohydrates can be moderately oxidized using reagents such as sodium periodate, and the resulting (-CHO) unit of oxidized carbohydrates can be combined with functional groups such as hydrazine, oxime, primary or secondary amines, hydrazine , Thiosemicarbazone, hydrazine carboxylate and aryl hydrazine), such as those described by Kaneko et al., 1991, Bioconjugate Chem. 2:133-41. The representative extension subunits of this embodiment are depicted in square brackets of formulas Via, VIb and VIc, where -R ¹⁷ -, L-, -W-, -Y-, -D, w and y are as defined above.

5.3.3.2 Amino acid unit

The amino acid unit (-W-) (when present) connects the extension subunit to the spacer unit (if the spacer unit is present), connects the extension subunit to the drug unit (if the spacer unit does not exist), and connects The antibody unit is connected to the drug unit (if the extension unit and the spacer unit are not present).

其中R¹⁹ 為氫、甲基、異丙基、異丁基、第二丁基、苯甲基、對羥基苯甲基、-CH₂ OH、-CH(OH)CH₃ 、-CH₂ CH₂ SCH₃ 、-CH₂ CONH₂ 、-CH₂ COOH、-CH₂ CH₂ CONH₂ 、-CH₂ CH₂ COOH、-(CH₂ )₃ NHC(=NH)NH₂ 、-(CH₂ )₃ NH₂ 、-(CH₂ )₃ NHCOCH₃ 、-(CH₂ )₃ NHCHO、-(CH₂ )₄ NHC(=NH)NH₂ 、-(CH₂ )₄ NH₂ 、-(CH₂ )₄ NHCOCH₃ 、-(CH₂ )₄ NHCHO、-(CH₂ )₃ NHCONH₂ 、-(CH₂ )₄ NHCONH₂ 、-CH₂ CH₂ CH(OH)CH₂ NH₂ 、2-吡啶基甲基-、3-吡啶基甲基-、4-吡啶基甲基-、苯基、環己基、

。W _w -can be, for example, a single peptide, dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, hepeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each -W- unit independently has the formula shown in square brackets below, and w is an integer in the range of 0 to 12:

Where R ¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl, p-hydroxybenzyl, -CH ₂ OH, -CH(OH)CH ₃ , -CH ₂ CH ₂ SCH ₃ , -CH ₂ CONH ₂ , -CH ₂ COOH, -CH ₂ CH ₂ CONH ₂ , -CH ₂ CH ₂ COOH, -(CH ₂ ) ₃ NHC(=NH)NH ₂ , -(CH ₂ ) ₃ NH ₂ , -(CH ₂ ) ₃ NHCOCH ₃ , -(CH ₂ ) ₃ NHCHO, -(CH ₂ ) ₄ NHC(=NH)NH ₂ , -(CH ₂ ) ₄ NH ₂ , -(CH ₂ ) ₄ NHCOCH ₃ , -(CH ₂ ) ₄ NHCHO, -(CH ₂ ) ₃ NHCONH ₂ , -(CH ₂ ) ₄ NHCONH ₂ , -CH ₂ CH ₂ CH(OH)CH ₂ NH ₂ , 2-pyridylmethyl-, 3 -Pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,

.

In some embodiments, the amino acid unit can be cleaved enzymatically by one or more enzymes (including cancer or tumor-related proteases) to release the drug unit (-D). In one embodiment, the drug unit is released in the living body. Protonation is carried out to obtain the drug (D).

其中R²⁰ 及R²¹ 為如下： R²⁰ R²¹ 苯甲基 (CH₂ )₄ NH₂ ； 甲基 (CH₂ )₄ NH₂ ； 異丙基 (CH₂ )₄ NH₂ ； 異丙基 (CH₂ )₃ NHCONH₂ ； 苯甲基 (CH₂ )₃ NHCONH₂ ； 異丁基 (CH₂ )₃ NHCONH₂ ； 第二丁基 (CH₂ )₃ NHCONH₂ ；

(CH₂ )₃ NHCONH₂ ； 苯甲基 甲基； 苯甲基 (CH₂ )₃ NHC(=NH)NH₂ ；

其中R²⁰ 、R²¹ 及R²² 為如下： R²⁰ R²¹ R²² 苯甲基 苯甲基 (CH₂ )₄ NH₂ ； 異丙基 苯甲基 (CH₂ )₄ NH₂ ；及 H 苯甲基 (CH₂ )₄ NH₂ ；

其中R²⁰ 、R²¹ 、R²² 及R²³ 為如下： R²⁰ R²¹ R²² R²³ H 苯甲基 異丁基 H；及 甲基 異丁基 甲基 異丁基。 In certain embodiments, the amino acid unit comprises a natural amino acid. In other embodiments, the amino acid unit comprises a non-natural amino acid. Illustrative Ww units are represented by the following formula VII to formula IX:

Where R ²⁰ and R ²¹ are as follows: R ²⁰ R ²¹ Benzyl (CH ₂ ) ₄ NH ₂ ; methyl (CH ₂ ) ₄ NH ₂ ; Isopropyl (CH ₂ ) ₄ NH ₂ ; Isopropyl (CH ₂ ) ₃ NHCONH ₂ ; Benzyl (CH ₂ ) ₃ NHCONH ₂ ; Isobutyl (CH ₂ ) ₃ NHCONH ₂ ; Second Butyl (CH ₂ ) ₃ NHCONH ₂ ;

(CH ₂ ) ₃ NHCONH ₂ ; Benzyl methyl; Benzyl (CH ₂ ) ₃ NHC(=NH)NH ₂ ;

Among them, R ²⁰ , R ²¹ and R ²² are as follows: R ²⁰ R ²¹ R ²² Benzyl Benzyl (CH ₂ ) ₄ NH ₂ ; Isopropyl Benzyl (CH ₂ ) ₄ NH ₂ ; and H Benzyl (CH ₂ ) ₄ NH ₂ ;

Among them, R ²⁰ , R ²¹ , R ²² and R ²³ are as follows: R ²⁰ R ²¹ R ²² R ²³ H Benzyl Isobutyl H; and methyl Isobutyl methyl Isobutyl.

Exemplary amino acid units include, but are not limited to, the units of formula VII above, wherein: R ²⁰ is benzyl and R ²¹ is -(CH ₂ ) ₄ NH ₂ ; R ²⁰ is isopropyl and R ²¹ is- (CH ₂ ) ₄ NH ₂ ; or R ²⁰ is isopropyl and R ²¹ is -(CH ₂ ) ₃ NHCONH ₂ .

Another exemplary amino acid unit is a unit of formula VIII, wherein R ²⁰ is a benzyl group, R ²¹ is a benzyl group, and R ²² is -(CH ₂ ) ₄ NH ₂ .

Suitable -W _w -units can be designed and optimized in terms of their selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases. In one embodiment, the -W _w -unit is a unit whose cleavage is catalyzed by cathepsin B, C and D or plasmin protease.

In one embodiment, -W _w -is a dipeptide, tripeptide, tetrapeptide or pentapeptide. When R ¹⁹ , R ²⁰ , R ²¹ , R ²² or R ^{23 are} not hydrogen, the carbon atom to which R ¹⁹ , R ²⁰ , R ²¹ , R ²² or R ²³ is connected is opposite.

Each carbon atom to which R ¹⁹ , R ²⁰ , R ²¹ , R ²² or R ²³ is connected is independently in (S) or (R) configuration.

In a specific embodiment, the amino acid unit is valine-citrulline (vc or Val-Cit). In another specific embodiment, the amino acid unit is phenylalanine-lysine (ie, fk). In yet another specific embodiment, the amino acid unit is N-methylvaline-citrulline. In yet another specific embodiment, the amino acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinoline carboxylate lysine, cyclohexylalanine lysine, isopiperidine Acid (isonepecotic acid) lysine, beta alanine lysine, glycine serine, valine, glutamic acid, and isonepecotic acid. 5.3.3.3 Spacer subunit

When the amino acid unit is present, the spacer unit (-Y-) (when present) connects the amino acid unit to the drug unit. Alternatively, when the amino acid unit is not present, the spacer unit connects the extension unit to the drug unit. When both the amino acid unit and the extension unit are absent, the spacer unit also connects the drug unit and the antibody unit.

There are two general types of spacer units: non-self-decomposing type or self-decomposing type. The non-self-decomposing spacer unit is a spacer unit in which part or all of the spacer unit remains bound to the drug unit after the amino acid unit is cleaved from the antibody drug conjugate (especially enzymatic cleavage). Examples of non-self-decomposing spacer units include (but are not limited to) (glycine-glycine) spacer units and glycine spacer units (both are described in Scheme 1) (see below). When the conjugate containing glycine-glycine spacer unit or glycine spacer unit is enzymatically cleaved by enzymes (such as tumor cell-related proteases, cancer cell-related proteases or lymphocyte-related proteases), glycine -Glycine-drug unit or glycine-drug unit is cleaved from L-Aa-Ww-. In one embodiment, the independent hydrolysis reaction occurs in the target cell, thereby breaking the bond of the glycine-drug unit and releasing the drug. Process 1

In some embodiments, the non-self-decomposing spacer unit (-Y-) is -Gly-. In some embodiments, the non-self-decomposing spacer unit (-Y-) is -Gly-Gly-.

In one embodiment, the spacer subunit does not exist (-Y _y -, where y=0).

Alternatively, an antibody-drug conjugate containing a self-decomposing spacer unit can release -D. As used herein, the term "self-decomposing spacer" refers to a bifunctional chemical moiety capable of covalently linking two spaced apart chemical moieties together into a stable tripartite molecule. If the bond with the first part is broken, it will spontaneously separate from the second chemical part.

In some embodiments, -Y _y -is a para-aminobenzyl alcohol (PAB) unit (see Schemes 2 and 3), the phenylene part of which is substituted with Q _m , where Q is -C ₁ -C ₈ alkyl, -C ₁ -C ₈ alkenyl, -C ₁ -C ₈ alkynyl, -O-(C ₁ -C ₈ alkyl), -O-(C ₁ -C ₈ alkenyl), -O-(C ₁ -C ₈ alkynyl), -halogen, -nitro or -cyano; and m is an integer in the range of 0-4. Alkyl, alkenyl, and alkynyl (whether alone or as part of another group) may optionally be substituted.

In some embodiments, -Y- is a PAB group, which is connected to -W _w -via the amino nitrogen atom of the PAB group, and directly connected to -D via a carbonate group, a urethane group or an ether group. connection. Without being bound by any particular theory or mechanism, Scheme 2 depicts the possible drug release mechanism of the PAB group directly connected to -D via a urethane or carbonate group, as described by Toki et al., 2002, J. Org. Chem. 67:1866-1872. Process 2

In Scheme 2, Q is -C ₁ -C ₈ alkyl, -C ₁ -C ₈ alkenyl, -C ₁ -C ₈ alkynyl, -O-(C ₁ -C ₈ alkyl), -O- (C ₁ -C ₈ alkenyl), -O-(C ₁ -C ₈ alkynyl), -halogen, -nitro or -cyano; m is an integer in the range of 0-4; and p is 1 to Within about 20. Alkyl, alkenyl, and alkynyl (whether alone or as part of another group) may optionally be substituted.

Without being bound by any particular theory or mechanism, Scheme 3 depicts a possible drug release mechanism of a PAB group directly connected to -D via an ether or amine linkage, where D includes an oxygen or nitrogen group that is part of the drug unit. Process 3

In Scheme 3, Q is -C ₁ -C ₈ alkyl, -C ₁ -C ₈ alkenyl, -C ₁ -C ₈ alkynyl, -O-(C ₁ -C ₈ alkyl), -O- (C ₁ -C ₈ alkenyl), -O-(C ₁ -C ₈ alkynyl), -halogen, -nitro or -cyano; m is an integer in the range of 0-4; and p is 1 to Within about 20. Alkyl, alkenyl, and alkynyl (whether alone or as part of another group) may optionally be substituted.

Other examples of self-decomposing spacers include, but are not limited to, aromatic compounds electronically similar to PAB groups, such as 2-aminoimidazole-5-methanol derivatives (Hay et al., 1999, Bioorg. Med. Chem . Lett. 9:2237) and o-aminobenzylacetaldehyde or p-aminobenzylacetaldehyde. Spacers that cyclize during the hydrolysis of the amide bond can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology 2:223), appropriately substituted bicyclic rings [2.2.1] and the bicyclic [2.2.2] ring system (Storm et al., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenyl propionate (Amsberry et al., 1990 , J. Org. Chem. 55:5867). The elimination of substituted amine-containing drugs at the alpha position of glycine (Kingsbury et al., 1984, J. Med. Chem. 27:1447) is also an example of a self-decomposing spacer.

In one embodiment, the spacer unit is a branched bis(hydroxymethyl)-styrene (BHMS) unit as depicted in Scheme 4, which can be used to incorporate and release multiple drugs. Process 4

In Scheme 4, Q is -C ₁ -C ₈ alkyl, -C ₁ -C ₈ alkenyl, -C ₁ -C ₈ alkynyl, -O-(C ₁ -C ₈ alkyl), -O- (C ₁ -C ₈ alkenyl), -O-(C ₁ -C ₈ alkynyl), -halogen, -nitro or -cyano; m is an integer in the range of 0-4; n is 0 or 1 ; And p is in the range of 1 to about 20. Alkyl, alkenyl, and alkynyl (whether alone or as part of another group) may optionally be substituted.

In some embodiments, the -D unit is the same. In yet another embodiment, the -D part is different.

其中Q為-C₁ -C₈ 烷基、-C₁ -C₈ 烯基、-C₁ -C₈ 炔基、-O-(C₁ -C₈ 烷基)、-O-(C₁ -C₈ 烯基)、-O-(C₁ -C₈ 炔基)、-鹵素、-硝基或-氰基；且m為在0-4範圍內之整數。烷基、烯基及炔基(無論單獨還是作為另一基團之一部分)可視情況經取代。

及

。In one aspect, the spacer subunit (-Y _y -) is represented by formulas X to XII :

Wherein Q is -C ₁ -C ₈ alkyl, -C ₁ -C ₈ alkenyl, -C ₁ -C ₈ alkynyl, -O-(C ₁ -C ₈ alkyl), -O-(C _1- C ₈ alkenyl), -O-(C ₁ -C ₈ alkynyl), -halogen, -nitro or -cyano; and m is an integer in the range of 0-4. Alkyl, alkenyl, and alkynyl (whether alone or as part of another group) may optionally be substituted.

and

.

其中w及y各自為0、1或2，及

其中w及y各自為0，

5.3.3.4 藥物負載 Examples of formula I and II including antibody-drug conjugate compounds may include:

Where w and y are each 0, 1, or 2, and

Where w and y are each 0,

5.3.3.4 Drug load

The drug load is represented by p, and is the average number of drug units in the molecule/antibody. The drug load can be in the range of 1 to 20 drug units (D)/antibody. The ADC provided herein includes a collection of antibodies or antigen-binding fragments that bind to a series of drug units (e.g., 1 to 20). In the preparation of ADC from the binding reaction, the average number of drug units/antibody can be characterized by conventional means such as mass spectrometry and ELISA analysis. The quantitative distribution of ADC in terms of p can also be determined. In some cases, the separation, purification, and characterization of a homogeneous ADC with a specific value of p from ADCs with other drug loadings can be achieved by means such as electrophoresis.

In certain embodiments, the drug loading of the ADC provided herein is in the range of 1-20. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1-18. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1-15. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1-12. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1-10. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1-9. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 8. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1-7. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 6. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 5. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 4. In certain embodiments, the drug loading of the ADC provided herein is in the range of 1 to 3. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2-12. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2-10. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2-9. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2-8. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2-7. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2-6. In certain embodiments, the drug loading of the ADC provided herein is in the range of 2 to 5.

In certain embodiments, the drug loading of the ADC provided herein is in the range of: 1 to about 8; about 2 to about 6; about 3 to about 5; about 3 to about 4; about 3.1 to about 3.9; about 3.2 to about 3.8; about 3.2 to about 3.7; about 3.2 to about 3.6; about 3.3 to about 3.8; or about 3.3 to about 3.7.

In certain embodiments, during the binding reaction, drug units less than the theoretical maximum are bound to the antibody. Antibodies may contain, for example, lysine residues that do not react with drug-linker intermediates or linker reagents. Generally speaking, antibodies do not contain many free and reactive cysteine thiol groups that can be linked to drug units; in fact, most of the cysteine thiol residues in antibodies exist in the form of disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethyl phosphine (TCEP) under partial or complete reduction conditions to generate reactive cysteine thiol groups. In certain embodiments, the antibody is subjected to denaturing conditions, exhibiting a reactive nucleophilic group, such as lysine or cysteine. In some embodiments, the linker unit or drug unit is bound via a lysine residue on the antibody unit. In some embodiments, the linker unit or drug unit is bound via a cysteine residue on the antibody unit.

In some embodiments, the amino acid connected to the linker unit drug unit is in the heavy chain of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid linked to the linker unit or the drug unit is in the light chain of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid connected to the linker unit or the drug unit is in the hinge region of the antibody or antigen-binding fragment thereof. In some embodiments, the amino acid connected to the linker unit or the drug unit is in the Fc region of the antibody or antigen-binding fragment thereof. In other embodiments, the amino acid connected to the linker unit or the drug unit is in the constant region of the antibody or antigen-binding fragment thereof (for example, CH1, CH2, or CH3 of the heavy chain, or CH1 of the light chain). In still other embodiments, the amino acid connected to the linker unit or the drug unit is in the VH framework region of the antibody or antigen-binding fragment thereof. In still other embodiments, the amino acid connected to the linker unit or the drug unit is in the VL framework region of the antibody or antigen-binding fragment thereof.

ADC loading (drug/antibody ratio) can be controlled in different ways, for example by: (i) limiting the drug-linker intermediate or linker reagent to the antibody molar excess, (ii) limiting the binding reaction time or temperature, (iii) Partial or restricted reduction conditions for the modification of cysteine thiol, (iv) engineering the amino acid sequence of the antibody by recombinant technology so that the number and position of cysteine residues are modified to Control the number and/or position of linker-drug linkages (such as thioMab or thioFab prepared as disclosed herein and WO2006/034488 (incorporated herein by reference in its entirety)).

It should be understood that more than one nucleophilic group reacts with the drug-linker intermediate or linker reagent, and then reacts with the drug unit reagent, and then the resulting product is the ADC compound connected to one or more drug units of the same antibody unit The distribution of the mixture. The average number of drugs/antibodies can be calculated from the mixture by double ELISA antibody analysis that is specific for antibodies and specific for drugs. Individual ADC molecules can be identified in the mixture by mass spectrometry and separated by HPLC (e.g., hydrophobic interaction chromatography) (see, e.g., Hamblett, KJ et al. "Effect of drug loading on the pharmacology, pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate", Abstract No. 624, American Association for Cancer Research, 2004 Annual Conference, March 27-31, 2004, AACR Conference Papers, Volume 45, March 2004; Alley, SC, etc. "Controlling the location of drug attachment in antibody-drug conjugates", Abstract No. 627, American Association for Cancer Research, 2004 Annual Conference, March 27-31, 2004, AACR Conference Paper, Volume 45, 2004 3 month). In some embodiments, a homogeneous ADC with a single loading value can be separated from the binding mixture by electrophoresis or chromatography. 5.3.3 Preparation of antibody-drug conjugate

The production of antibody-drug conjugates provided herein can be achieved by any technique known to those skilled in the art. In short, an antibody-drug conjugate includes an anti-191P4D12 antibody or an antigen-binding fragment thereof as an antibody unit, a drug, and a linker that connects the drug and the binding agent as appropriate. In some embodiments, the antibody is an anti-191P4D12 antibody comprising the CDR region of the antibody called Ha22-2(2,4)6.1 described above. In a specific embodiment, the antibody is an anti-191P4D12 antibody comprising the heavy chain and light chain variable regions of the antibody named Ha22-2(2,4)6.1 described above. In a specific embodiment, the antibody is an anti-191P4D12 antibody comprising the heavy and light chains of the antibody named Ha22-2(2,4)6.1 described above.

A variety of different reactions can be used for the covalent attachment of drugs and/or linkers to binding agents. This usually depends on the amino acid residues of the binding agent (such as antibody molecules) (including the amine groups of lysine, the free carboxylic acid groups of glutamine and aspartic acid, the sulfhydryl groups of cysteine and the aromatic The reaction of multiple parts of the family amino acid) is realized. One of the most commonly used non-specific methods for covalent attachment is to react the carboxyl group (or amine group) of the compound with the carbodiimide group of the antibody. In addition, bifunctional agents such as dialdehydes or imidates have been used to link the amine groups of compounds to the amine groups of antibody molecules. The Schiff base reaction can also be used to link the drug to the binding agent. This method involves the oxidation of the periodate of drugs containing glycols or hydroxyl groups, thereby forming aldehydes which subsequently react with the binding agent. The connection occurs via the formation of a Schiff base with the amine group of the binding agent. Isothiocyanates can also be used as coupling agents for covalently linking drugs and binding agents. Other technologies are known to those skilled in the art and are within the scope of the present invention.

In certain embodiments, the intermediate (which is the linker precursor) reacts with the drug under appropriate conditions. In some embodiments, reactive groups on drugs and/or intermediates are used. The product of the reaction between the drug and the intermediate or the derivative drug then reacts with the anti-191P4D12 antibody under appropriate conditions.

Each of the specific units of the antibody drug conjugate is described in more detail herein. The synthesis and structure of exemplary linker units, extension units, amino acid units, self-decomposing spacer units and drug units are also described in U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649 and 2005 -0009751, each of which is incorporated herein by reference in its entirety and for all purposes.

Exemplary methods for producing the antibody drug conjugates provided herein are briefly described below.

其中：    AA1 =胺基酸1 AA2 =胺基酸2 AA5 =胺基酸5 DIL = 多拉索因(Dolaisoleuine) DAP = 多拉普因(Dolaproine) 連接子= Val-Cit (vc)Using the following scheme, using the vc (Val-Cit) linker described herein, the Ha22-2(2,4)6.1 antibody was combined with the auristatin derivative MMAE to produce an antibody-drug conjugate (ADC) (called AGS) -22M6E). The general method described in the following scheme 5 was used to complete the combination of vc (Val-Cit) linker and MMAE (Seattle Genetics, Inc., Seattle, WA) to produce cytotoxic vcMMAE (see US Patent No. 7,659,241). Process 5 General method for synthesis of vcMMAE

Among them: AA1 = amino acid 1 AA2 = amino acid 2 AA5 = amino acid 5 DIL = Dolaisoleuine DAP = Dolaproine Linker = Val-Cit (vc)

Next, the following protocol was used to prepare the antibody drug conjugate AGS-22M6E.

In short, add 15 mg/mL solution of Ha22-2(2,4)6.1 antibody in 10 mM acetate pH 5.0, 1% sorbitol, 3% L-arginine, and add 20% volume 0.1 M TrisCl pH 8.4, 25mM EDTA and 750 mM NaCl to adjust the pH of the solution to 7.5, 5mM EDTA and 150 mM sodium chloride. Then, the antibody was partially reduced by adding 2.3 mole equivalents of TCEP (the number of moles relative to MAb), and then stirring was carried out at 37°C for 2 hours. Then, the partially reduced antibody solution was cooled to 5°C, and 4.4 mole equivalents of vcMMAE (relative to the number of moles of antibody) in the form of 6% (v/v) DMSO solution were added. The mixture was stirred at 5°C for 60 minutes, and then stirred for an additional 15 minutes after adding 1 molar equivalent of N-acetylcysteine relative to vcMMAE. Ultrafiltration/diafiltration antibody drug conjugate (ADC) and 10 volumes of 20 mM histidine pH 6.0 were used to remove excess quenched vcMMAE and other reaction components.

其中L為Ha22-2(2,4)6.1，且p為1至20。5.4 使用醫藥組合物之方法 The resulting antibody drug conjugate AGS-22M6E has the following formula:

Where L is Ha22-2(2,4)6.1, and p is 1-20. 5.4 Methods of using pharmaceutical compositions

In one aspect, provided herein is a method of preventing or treating a disease or condition in an individual, which comprises administering to the individual an effective amount of the pharmaceutical composition provided herein. In some embodiments, the individual is a human individual.

In some embodiments, the disease or condition is cancer. In some embodiments, the cancer has tumor cells expressing 191P4D12. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is colon cancer, pancreatic cancer, ovarian cancer, lung cancer, bladder cancer, breast cancer, esophageal cancer, head cancer, or neck cancer. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is advanced bladder cancer. In some embodiments, the cancer is metastatic bladder cancer. In some embodiments, the cancer is urothelial carcinoma. In some embodiments, the cancer is advanced urothelial carcinoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is head cancer. In some embodiments, the cancer is neck cancer. In some embodiments, the cancer is advanced or metastatic cancer.

In some embodiments, the instruction is to treat an individual who has received one or more rounds of chemotherapy with the pharmaceutical composition provided herein. Alternatively, for individuals not receiving chemotherapy treatment, the pharmaceutical composition provided herein is combined with chemotherapy or radiation regimens. In addition, in some embodiments, the use of the pharmaceutical compositions provided herein may enable the use of reduced dose concurrent chemotherapy, in particular for individuals who are not very resistant to the toxicity of chemotherapeutic agents. In some embodiments, the pharmaceutical composition disclosed herein is administered to patients with metastatic urothelial carcinoma who have shown disease progression or recurrence during or after treatment with immune checkpoint inhibitors.

Methods of administering the pharmaceutical compositions provided herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural and transmucosal (e.g. intranasal and Oral route). In a specific embodiment, the pharmaceutical composition provided herein is administered intranasally, intramuscularly, intravenously, or subcutaneously. The pharmaceutical compositions provided herein can be administered by any convenient route, such as by infusion or rapid injection, by transepithelial or mucosal skin lining (for example, oral mucosa, rectum and intestinal mucosa, etc.) absorption, and can be combined with Other biologically active agents are administered together. Administration can be systemic or local. In addition, pulmonary administration can also be used, for example, an inhaler or nebulizer, and a formulation with aerosol. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publications WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety.

In a specific embodiment, it may be necessary to locally administer the pharmaceutical composition provided herein to the area in need of treatment. This can be achieved by, for example, but not as a limitation: local infusion, local administration (for example by intranasal spray), injection, or by means of an implant which is porous, non-porous or glued Shaped materials include membranes, such as silicone rubber membranes, or fibers. In some embodiments, when administering the pharmaceutical compositions provided herein, care must be taken to use materials that are not absorbed by the antibody-drug conjugate provided herein.

In another embodiment, the pharmaceutical composition provided herein can be delivered in vesicles, especially liposomes (see Langer, 1990, Science 249:1527-1533; Treat et al., Liposomes in the Therapy of Infectious Disease and Cancer , Lopez-Berestein and Fidler (eds), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In another embodiment, the pharmaceutical composition provided herein can be delivered in a controlled release or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve the controlled release or sustained release of the prophylactic or therapeutic agents provided herein (such as the antibody-drug conjugate provided herein) or the pharmaceutical composition (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); US Patent No. 5,679,377; US Patent No. 5,916,597; US Patent No. 5,912,015; US Patent No. 5,989,463; US Patent No. 5,128,326; PCT Publication No. WO 99/15154; And PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include (but are not limited to) poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-acetic acid) Vinyl ester), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polypropylene amide, poly(ethylene glycol), Polylactide (PLA), poly(lactide-co-glycolide) (PLGA) and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of percolable impurities, stable in storage, sterile, and biodegradable. In yet another embodiment, a controlled release or sustained release system can be placed near the treatment target (ie, nasal passage or lung), so that only a part of the systemic dose is required (see, for example, Goodson, Medical Applications of Controlled Release, See above, Vol. 2, pp. 115-138 (1984)). The controlled release system is discussed in Langer's review (1990, Science 249:1527-1533). Any technique known to those skilled in the art can be used to produce sustained release formulations comprising the antibody drug conjugates or pharmaceutical compositions provided herein. See, eg, US Patent No. 4,526,938; PCT Publication WO 91/05548; PCT Publication WO 96/20698; Ning et al., 1996, "Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel", Radiotherapy & Oncology 39:179-189; Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application", Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery", Proc. Int 'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of these documents is incorporated herein by reference in its entirety.

The amount of the pharmaceutical composition provided herein that will be effective in preventing and/or treating cancer can be determined by standard clinical techniques. In addition, in vitro analysis can be used to help identify the best dose range depending on the situation. The precise dose to be used will also depend on the route of administration and the severity of the disease or condition, and should be determined based on the judgment of the practitioner and individual conditions. The effective dose can be extrapolated from the dose response curve derived from in vitro or animal model test systems.

In certain embodiments, the treatment methods provided herein encompass the administration of a single ADC and a combination, or a mixture of different ADCs comprising different anti-191P4D12 antibodies or different drug units. In some embodiments, such methods have certain advantages because, for example, they contain ADCs that target different epitopes, utilize different effect mechanisms or directly combine cytotoxic antibodies with antibodies that depend on immune effector functionality. Such methods can exhibit synergistic therapeutic effects. In addition, the pharmaceutical composition provided herein can be administered simultaneously with other treatment modalities including (but not limited to) various chemotherapeutics and biological agents, androgen blockers, immunomodulators (such as IL- 2. GM-CSF), surgery or radiation.

In one embodiment, there is a synergistic effect when the pharmaceutical composition provided herein is used in combination with chemotherapeutic agents or radiation or a combination thereof to treat tumors (including human tumors).

The method for inhibiting the growth of tumor cells using the pharmaceutical composition provided herein and chemotherapy or radiation or a combination of both includes before, during or after starting chemotherapy or radiation therapy and any combination thereof (that is, in Before and during the start of chemotherapy and/or radiation therapy, before and after, during and after, or before, during and after), the pharmaceutical composition of the present invention is administered. Depending on the treatment plan and the needs of the specific patient, the method is performed in a way that will provide the most effective treatment and ultimately prolong the life of the patient.

The administration of chemotherapeutic agents can be achieved in a variety of ways, including by systemic parenteral and enteral routes. In one embodiment, the chemotherapeutic agent is administered separately. Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), actinomycin, methyl bis(chloroethyl) amine (nitrogen mustard), streptozotocin, cyclin Phosphoamide, carmustine (BCNU), lomustine (CCNU), cranberries (adriamycin), daunorubicin, procarbazine Hydrazine (procarbazine), mitomycin, cytarabine, etoposide (etoposide), methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin (bleomycin), paclitaxel (paclitaxel) ), docetaxel (taxane), aldesleukin, aspartase, busulfan (busulfan), carboplatin, cladribine, dacarbazine ( dacarbazine), fluorouridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol, melphalan , Mercaptopurine, mercaptopurine, plicamycin, mitotane, pegaaspargase, pentostatin, pipobroman, pracamycin ( plicamycin), streptozotocin, tamoxifen, teniposide, testosterone, thioguanine, thiotepa, uracil mustard, vinorelbine , Gemcitabine, chlorambucil, paclitaxel and combinations thereof.

The radiation sources used in combination with the pharmaceutical compositions provided herein can be external or internal to the patient being treated. When the source is outside the patient, the therapy is called external beam radiation therapy (EBRT). When the radiation source is inside the patient, the treatment is called brachytherapy (BT).

The treatment regimens described above can be additionally combined with additional cancer therapeutic agents and/or regimens, such as additional chemotherapy, cancer vaccines, signal transduction inhibitors, agents suitable for the treatment of abnormal cell growth or cancer, antibodies (such as WO /2005/092380 (Pfizer) described in anti-CTLA-4 antibody) or other ligands that inhibit tumor growth by binding to IGF-1R, and cytokines.

When the mammal is subjected to additional chemotherapy, the chemotherapeutic agents described above can be used. In addition, growth factor inhibitors, biological response modifiers, antihormonal therapy, selective estrogen receptor modulators (SERM), angiogenesis inhibitors, and antiandrogens can be used. For example, anti-hormones can be used, such as anti-estrogens (such as Nolvadex (tamoxifen)) or anti-androgens (such as Casodex (4'-cyano- 3-(4-Fluorophenylsulfonyl)-2-hydroxy-2-methyl-3-(trifluoromethyl)propananiline)).

In some embodiments, the drugs provided herein are used in combination with a second therapeutic agent, for example to treat cancer.

In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that reduces, inhibits, interferes with, or modulates one or more checkpoint proteins in whole or in part. Without wishing to be bound by any particular theory, checkpoint proteins regulate T cell activation or function. A variety of checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer , 2012 , 12 , 252-264) . These proteins seem to be responsible for costimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins seem to regulate and maintain self-tolerance and the duration and magnitude of physiological immune responses. Immune checkpoint inhibitors include antibodies or are derived from antibodies.

In one embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is an anti-CTLA-4 antibody. Examples of anti-CTLA-4 antibodies include, but are not limited to, those described in: U.S. Patent Nos. 5,811,097, 5,811,097, 5,855,887, 6,051,227, 6,207,157, 6,682,736, 6,984,720 No. and No. 7,605,238, these patents are all incorporated by reference in their entirety. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (also known as ticilimumab or CP-675,206). In another embodiment, the anti-CTLA-4 antibody is ipilimumab (also known as MDX-010 or MDX-101). Ipilimumab is a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is sold under the trade name Yervoy™.

In one embodiment, the checkpoint inhibitor is a PD-1/PD-L1 inhibitor. Examples of PD-L/PD-L1 inhibitors include, but are not limited to, those described in: U.S. Patent Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, and PCT Patents Application Publication Nos. WO2003042402, WO2008156712, WO2010089411, WO2010036959, WO2011066342, WO2011159877, WO2011082400, and WO2011161699, all of which are incorporated herein by reference in their entirety .

In one embodiment, the checkpoint inhibitor is a PD-1 inhibitor. In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody. In one embodiment, the anti-PD-1 antibody is BGB-A317, nivolumab (also known as ONO-4538, BMS-936558 or MDX1106) or pembrolizumab (also known as MK-3475, SCH 900475 or lambrolizumab). In one embodiment, the anti-PD-1 antibody is nivolumab. Nivolumab is a human IgG4 anti-PD-1 monoclonal antibody and is sold under the brand name Opdivo™. In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 antibody and is sold under the brand name Keytruda™. In yet another embodiment, the anti-PD-1 antibody is the humanized antibody CT-011. In the case of recurrence, CT-011 administered alone failed to show response to the treatment of acute myeloid leukemia (AML). In yet another embodiment, the anti-PD-1 antibody is the fusion protein AMP-224. In another embodiment, the PD-1 antibody is BGB-A317. BGB A317 is a monoclonal antibody, which specifically engineered the ability to bind to Fc γ receptor I, and it has the characteristics of uniquely binding to PD-1 with high affinity and excellent target specificity.

In one embodiment, the checkpoint inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody. In one embodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab). In another embodiment, the anti-PD-L1 antibody is BMS-936559 (also known as MDX-1105-01). In yet another embodiment, the PD-L1 inhibitor is atezolizumab (also known as MPDL3280A and Tecentriq®).

In one embodiment, the checkpoint inhibitor is a PD-L2 inhibitor. In one embodiment, the PD-L2 inhibitor is an anti-PD-L2 antibody. In one embodiment, the anti-PD-L2 antibody is rHIgM12B7A.

In one embodiment, the checkpoint inhibitor is a lymphocyte activation gene-3 (LAG-3) inhibitor. In one embodiment, the LAG-3 inhibitor is the soluble Ig fusion protein IMP321 (Brignone et al., J. Immunol. , 2007 , 179 , 4202-4211). In another embodiment, the LAG-3 inhibitor is BMS-986016.

In one embodiment, the checkpoint inhibitor is a B7 inhibitor. In one embodiment, the B7 inhibitor is a B7-H3 inhibitor or a B7-H4 inhibitor. In one embodiment, the B7-H3 inhibitor is the anti-B7-H3 antibody MGA271 (Loo et al., Clin. Cancer Res. , 2012 , 3834).

In one embodiment, the checkpoint inhibitor is a TIM3 (T cell immunoglobulin domain and mucin domain 3) inhibitor (Fourcade et al., J. Exp. Med. , 2010 , 207 , 2175-86; Sakuishi et al. , J. Exp. Med. , 2010 , 207 , 2187-94).

In one embodiment, the checkpoint inhibitor is an OX40 (CD134) agonist. In one embodiment, the checkpoint inhibitor anti-OX40 antibody. In one embodiment, the anti-OX40 antibody is anti-OX-40. In another embodiment, the anti-OX40 antibody is MEDI6469.

In one embodiment, the checkpoint inhibitor is a GITR agonist. In one embodiment, the checkpoint inhibitor anti-GITR antibody. In one embodiment, the anti-GITR antibody is TRX518.

In one embodiment, the checkpoint inhibitor is a CD137 agonist. In one embodiment, the checkpoint inhibitor anti-CD137 antibody. In one embodiment, the anti-CD137 antibody is urelumab. In another embodiment, the anti-CD137 antibody is PF-05082566.

In one embodiment, the checkpoint inhibitor is a CD40 agonist. In one embodiment, the checkpoint inhibitor is an anti-CD40 antibody. In one embodiment, the anti-CD40 antibody is CF-870,893.

In one embodiment, the checkpoint inhibitor is recombinant human interleukin-15 (rhIL-15).

In one embodiment, the checkpoint inhibitor is an IDO inhibitor. In one embodiment, the IDO inhibitor is INCB024360. In another embodiment, the IDO inhibitor is indoximod.

In certain embodiments, the combination therapy provided herein includes two or more of the checkpoint inhibitors described herein (including checkpoint inhibitors of the same or different categories). In addition, the combination therapies described herein can be used in combination with one or more second active agents as described herein as appropriate to treat diseases described herein and understood in the art.

In some embodiments, the checkpoint inhibitor is administered before the pharmaceutical composition of the invention is administered. In other embodiments, the checkpoint inhibitor is administered at the same time as the pharmaceutical composition provided herein (eg, in the same administration period). In yet other embodiments, the checkpoint inhibitor is administered after the pharmaceutical composition provided herein is administered.

In some embodiments, the amount of checkpoint inhibitor can be determined by standard clinical techniques.

The dose of the checkpoint inhibitor produces a serum titer of about 0.1 μg/ml to about 450 μg/ml, and in some embodiments, the following doses can be administered to humans to prevent and/or treat cancer: at least 0.1 μg/ml , At least 0.2 μg/ml, at least 0.4 μg/ml, at least 0.5 μg/ml, at least 0.6 μg/ml, at least 0.8 μg/ml, at least 1 μg/ml, at least 1.5 μg/ml, such as at least 2 μg/ml, At least 5 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 30 μg/ml, at least 35 μg/ml, at least 40 μg/ml, at least 50 μg/ml, at least 75 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 200 μg/ml, at least 250 μg/ml, at least 300 μg/ml, at least 350 μg/ ml, at least 400 μg/ml, or at least 450 μg/ml. It should be understood that the precise dose of the checkpoint inhibitor to be used will also depend on the route of administration and the severity of the individual's cancer, and should be determined based on the judgment of the practitioner and the condition of each patient.

In some embodiments, the dose of the checkpoint inhibitor (eg, PD-1 inhibitor or PD-L1 inhibitor) administered to the patient is usually 0.1 mg/kg to 100 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is from about 1 mg/kg to about 75 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is between 1 mg/kg and 20 mg/kg individual body weight, such as 1 mg/kg to 5 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 1 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 1.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 2 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 2.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 3 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 3.5 mg/kg of individual body weight. In some embodiments, the dose administered to the patient is about 4 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 4.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 5.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 6 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 6.5 mg/kg body weight. In some embodiments, the dose administered to the patient is about 7 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 7.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 8 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 8.5 mg/kg of individual body weight. In some embodiments, the dose administered to the patient is about 9.0 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 10.0 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 15.0 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 20.0 mg/kg of the individual's body weight.

In some embodiments, the pharmaceutical composition provided herein is supplied in the form of a dry sterile lyophilized powder or an anhydrous concentrate in an airtight sealed container, and can be reconstituted with, for example, water or saline to an appropriate concentration for administration to an individual . In certain embodiments, the antibody-drug conjugate is supplied as a dry sterile lyophilized powder in an airtight sealed container in a unit dose of at least 0.1 mg, at least 0.5 mg, at least 1 mg, at least 2 mg, or at least 3 mg, such as at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 60 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg. The lyophilized antibody-drug conjugate can be stored in its initial container between 2 and 8°C, and the antibody-drug conjugate can be within 12 hours after recovery, such as within 6 hours, within 5 hours, within 3 hours, or within 1 hour Vote. In an alternative embodiment, the pharmaceutical composition comprising the antibody-drug conjugate provided herein is supplied in liquid form in an airtight container, which indicates the amount and concentration of the antibody-drug conjugate. In certain embodiments, the antibody-drug conjugate in liquid form is supplied in an air-tight container at the following doses: at least 0.1 mg/ml, at least 0.5 mg/ml, or at least 1 mg/ml, such as at least 5 mg /ml, at least 10 mg/ml, at least 15 mg/ml, at least 25 mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least 60 mg/ml, at least 70 mg/ml , At least 80 mg/ml, at least 90 mg/ml or at least 100 mg/ml.

In some embodiments, the amount of prophylactic or therapeutic agents (such as the antibody drug conjugate provided herein) or the pharmaceutical composition provided herein that will be effective in preventing and/or treating cancer can be determined by standard clinical techniques .

Therefore, the dosage of the antibody-drug conjugate in the pharmaceutical composition produces a serum titer of about 0.1 μg/ml to about 450 μg/ml, and in some embodiments, the following dosages can be administered to humans for prevention and/or treatment Cancer: at least 0.1 μg/ml, at least 0.2 μg/ml, at least 0.4 μg/ml, at least 0.5 μg/ml, at least 0.6 μg/ml, at least 0.8 μg/ml, at least 1 μg/ml, at least 1.5 μg/ml, Such as at least 2 μg/ml, at least 5 μg/ml, at least 10 μg/ml, at least 15 μg/ml, at least 20 μg/ml, at least 25 μg/ml, at least 30 μg/ml, at least 35 μg/ml, at least 40 μg/ml, at least 50 μg/ml, at least 75 μg/ml, at least 100 μg/ml, at least 125 μg/ml, at least 150 μg/ml, at least 200 μg/ml, at least 250 μg/ml, at least 300 μg /ml, at least 350 μg/ml, at least 400 μg/ml, or at least 450 μg/ml. It should be understood that the precise dosage to be used in the formulation will also depend on the route of administration and the severity of the individual's cancer, and should be determined based on the judgment of the practitioner and the condition of each patient.

The effective dose can be extrapolated from the dose response curve derived from in vitro or animal model test systems.

For a pharmaceutical composition comprising the antibody-drug conjugate provided herein, the dose of the antibody-drug conjugate administered to the patient is usually 0.1 mg/kg to 100 mg/kg of the body weight. In some embodiments, the dose administered to the patient is from about 1 mg/kg to about 75 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is between 1 mg/kg and 20 mg/kg individual body weight, such as 1 mg/kg to 5 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 1 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 1.25 mg/kg of individual body weight. In some embodiments, the dose administered to the patient is about 1.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 2 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 2.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 3 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 3.5 mg/kg of individual body weight. In some embodiments, the dose administered to the patient is about 4 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 4.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 5.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 6 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 6.5 mg/kg body weight. In some embodiments, the dose administered to the patient is about 7 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 7.5 mg/kg of the individual's body weight. In some embodiments, the dose administered to the patient is about 8 mg/kg individual body weight. In some embodiments, the dose administered to the patient is about 8.5 mg/kg of individual body weight.

In some embodiments, the antibody drug conjugate formulated in the pharmaceutical composition provided herein is administered based on the patient's actual body weight at baseline, and unless the patient's body weight changes by ≥10% from the baseline of the previous cycle or meets the dose Adjust the standard, otherwise the dose will remain unchanged. In some embodiments, the actual weight will be used unless the patient weighs more than 100 kg, in which case the dose will be calculated based on a weight of 100 kg. In some embodiments, for patients receiving the 1.00 mg/kg dose level, the maximum dose is 100 mg; and for patients receiving the 1.25 mg/kg dose level, the maximum dose is 125 mg.

In one embodiment, the following amount of antibody-drug conjugate formulated in the pharmaceutical composition of the present invention is administered 5 times, 4 times, 3 times, 2 times or once to treat cancer: approximately 100 mg/kg or less , Approximately 75 mg/kg or less, approximately 50 mg/kg or less, approximately 25 mg/kg or less, approximately 10 mg/kg or less, approximately 5 mg/kg or less, approximately 1 mg/ kg or less, approximately 0.5 mg/kg or less, or approximately 0.1 mg/kg or less. In some embodiments, the pharmaceutical composition comprising the antibody-drug conjugate provided herein is administered about 1-12 times, wherein the doses may be required, such as once a week, once every two weeks, once a month, once every two months, Wait for the administration once in March, as determined by the physician. In some embodiments, lower doses (e.g., 0.1-15 mg/kg) may be administered more frequently (e.g., 3-6 times). In other embodiments, higher doses (e.g., 25-100 mg/kg) may be administered infrequently (e.g., 1-3 times).

In some embodiments, for each two-week cycle (for example, about 14 days) for a certain period of time (for example, one year), a single dose of the antibody-drug conjugate formulated in the pharmaceutical composition provided herein is administered to the patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 times to prevent and/or treat cancer, where the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, About 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg or a combination thereof (ie, Each monthly dose may be the same or different).

In some embodiments, for each three-week period (for example, about 21 days) for a certain period of time (for example, one year), a single dose of the antibody-drug conjugate formulated in the pharmaceutical composition provided herein is administered to the patient 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 times to prevent and/or treat cancer, where the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, About 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg or a combination thereof (ie, Each monthly dose may be the same or different).

In some embodiments, for every four-week period (for example, about 28 days) for a certain period of time (for example, one year), a single dose of the antibody-drug conjugate formulated in the pharmaceutical composition provided herein is administered to the patient 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 To prevent and/or treat cancer, the dosage is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg /kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg , About 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg or a combination thereof (that is, every A monthly dose may be the same or different).

In another embodiment, a single dose of the antibody-drug conjugate formulated in the pharmaceutical composition provided herein is administered to the patient at intervals of about one month (for example, about 30 days) for a certain period of time (for example, one year) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times to prevent and/or treat cancer, wherein the dosage is selected from the group consisting of: about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, About 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg, or a combination thereof (that is, each monthly dose may be the same or may be different).

In another embodiment, a single dose of the antibody-drug combination formulated in the pharmaceutical composition provided herein is administered to the patient at intervals of about two months (for example, about 60 days) for a certain period of time (for example, one year) 1, 2, 3, 4, 5 or 6 times to prevent and/or treat cancer, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg /kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg , About 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg or a combination thereof (that is, each monthly dose may be the same or may be different).

In yet another embodiment, a single dose of the antibody drug formulated in the pharmaceutical composition provided herein is administered to the patient at intervals of about three months (for example, about 120 days) for a certain period of time (for example, one year) Combine 1, 2, 3 or 4 times to prevent and/or treat cancer, wherein the dose is selected from the group consisting of about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.25 mg/kg kg, about 1.5 mg/kg, about 2 mg/kg, about 2.5 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, About 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, about 100 mg/kg kg or a combination thereof (that is, each monthly dose may be the same or may be different).

In certain embodiments, the route of administration for administering to the patient the dosage of the antibody drug conjugate formulated in the pharmaceutical composition provided herein is intranasal, intramuscular, intravenous, or a combination thereof, but as described herein The other approaches described are also acceptable. Each dose may or may not be administered by the same route of administration. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical compositions provided herein can be administered simultaneously or subsequently with other dosages of one or more additional therapeutic agents via multiple administration routes.

In some more specific embodiments, the antibody drug conjugate formulated in the pharmaceutical composition provided herein is administered intravenously at a dose of about 1 mg/kg, about 1.25 mg/kg, or about 1.5 mg/kg of the individual’s body weight. Intravenous (IV) injection or infusion administration.

In some more specific embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition provided herein is at a dose of about 1 mg/kg, 1.25 mg/kg or about 1.5 mg/kg of the individual’s body weight, by every three Two intravenous (IV) injections or infusions were administered in approximately 30 minutes every week. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion for about 30 minutes on the first and eighth days of every three-week cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion one or more times in each three-week cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion on day 1 of every three-week cycle. In some embodiments, the immune checkpoint inhibitor is pembrolizumab, and pembrolizumab is administered in an amount of about 200 mg over about 30 minutes. In other embodiments, the immune checkpoint inhibitor is atezolizumab, and wherein atezolizumab is administered in an amount of about 1200 mg over about 60 minutes or 30 minutes. In some embodiments, an antibody-drug conjugate is administered to patients with urothelial cancer who have shown disease progression or recurrence during or after treatment with immune checkpoint inhibitors. In some embodiments, the antibody-drug conjugate is administered to patients with metastatic urothelial carcinoma who have shown disease progression or recurrence during or after treatment with immune checkpoint inhibitors.

In other more specific embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition provided herein is at a dose of about 1 mg/kg, 1.25 mg/kg, or about 1.5 mg/kg of the individual’s body weight. Three intravenous (IV) injections or infusions for about 30 minutes were administered in a four-week cycle. In some embodiments, the antibody-drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion for about 30 minutes on days 1, 8 and 15 of every four-week cycle. In some embodiments, the method further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion one or more times in each four-week cycle. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In other embodiments, the immune checkpoint inhibitor is atezolizumab. In some embodiments, an antibody-drug conjugate is administered to patients with urothelial cancer who have shown disease progression or recurrence during or after treatment with immune checkpoint inhibitors. In some embodiments, the antibody-drug conjugate is administered to patients with metastatic urothelial carcinoma who have shown disease progression or recurrence during or after treatment with immune checkpoint inhibitors.

For the sake of brevity, certain abbreviations are used in this article. One example is a one-letter abbreviation for amino acid residues. Amino acids and their corresponding three-letter and one-letter abbreviations are as follows: Alanine Ala (A) Arginine Arg (R) Asparagine Asn (N) Aspartic acid Asp (D) Cysteine Cys (C) Glutamate Glu (E) Glutamic acid Gln (Q) Glycine Gly (G) Histidine His (H) Isoleucine Ile (I) Leucine Leu (L) Lysine Lys (K) Methionine Met (M) Phenylalanine Phe (F) Proline Pro (P) Serine Ser (S) Threonine Thr (T) Tryptophan Trp (W) Tyrosine Tyr (Y) Valine Val (V)

Herein, the present invention is generally disclosed by describing multiple embodiments using positive language. The present invention also specifically includes embodiments in which specific subjects are completely or partially excluded, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis. Therefore, although the present invention is not generally described in terms of what is not included in the present invention, aspects that are not explicitly included in the present invention are disclosed herein.

Specific embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. After reading the foregoing description, variants of the disclosed embodiments may become obvious to individuals working in this technology, and we expect that those familiar with this technology can adopt such variants as needed. Therefore, it is intended that the present invention is different from being practiced as specifically described herein, and the present invention includes all modifications and equivalents of the subject matter described in the scope of the appended application as permitted by applicable law. Furthermore, unless otherwise indicated herein or otherwise clearly contradictory to the context, the present invention encompasses any combination of the above-described elements in all possible variations thereof.

All publications, patent applications, deposit numbers and other references cited in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent application is specifically and individually indicated as being cited The way is merged into the general. The public case discussed in this article only provides the disclosure content before the filing date of this application. This article should not be construed as an admission that the present invention has no right to precede such disclosures by means of previous inventions. In addition, the date of the public case provided may be different from the date of the actual public case that may require independent confirmation.

Many embodiments of the invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the description in the experimental section is intended to illustrate rather than limit the scope of the invention described in the scope of the patent application. 6. Examples

The following is a description of various methods and materials used in the test, and is proposed to provide a complete disclosure and description of how to prepare and use the present invention to those skilled in the art, and is not intended to limit the inventors to regard as the content of the invention. The scope of the content, and it is not intended to indicate that the following experiments have been carried out and are all available experiments. It should be understood that the illustrative descriptions written in the present tense are not necessarily performed, but can be performed to generate information related to the teachings of the present invention and the like. Efforts have been made to ensure the accuracy of the number used (such as amount, temperature, etc.), but some experimental errors and deviations should be considered. 6.1 Example 1 - pH and buffer screening

AGS-22M6E was formulated at 10 mg/mL in fourteen candidate buffers (all at 20 mM, as detailed in Table 1 below). The evaluation used 20 mM sodium citrate buffer, titrated with citric acid to pH 5.2 and 5.7 formulations, and the evaluation used 20 mM histidine buffer, titrated with HCl to pH 5.5, 6.0, and 6.5 formulations. In addition, in the histidine buffer system, three different anions are evaluated: chloride, phosphate and succinate. The liquid formulation was subjected to 40°C storage temperature conditions for 2 weeks, room temperature (RT) agitation for 24 hours, and freeze-thaw cycles (frozen at -70°C and thawed at 20°C-25°C for 1, 3, and 10 cycles). Table 1 Formulation number Buffer pH Trehalose dihydrate (%) Sucrose (%) Tween 20 concentration (w/v%) F1 20 mM sodium citrate / citric acid 5.2 5.5 0 0.02 F2 5.7 F3 20 mM histidine / HC1 5.5 F4 6 F5 6.5 F6 20 mM sodium citrate / citric acid 5.2 0 5 F7 5.7 F8 20 mM histidine / HC1 5.5 F9 6 F10 6.5 F11 20 mM histidine / phosphoric acid 5.5 5.5 0 F12 6 F13 20 mM histidine / succinic acid 5.5 F14 6 Note : 5% sucrose (m. wt. 342) = 146 mM; 5.5% trehalose dihydrate (m. wt. 378) = 146 mM.

The formulation preparation and experimental design are described in more detail below. Protein product used for formulation test

Four test tubes each containing about 50 ml of AGS-22M6E (batch number AGS22M6-VCE-02) totaling about 2.5 grams were frozen. AGS-22M6E is 12.5 mg/mL in 20 mM histidine pH 6.0 buffer containing 5% sucrose and 0.02% polysorbate-20. Store the material at -70°C until use. Preparation of formulation buffer

According to the following table 2, the preparation includes citric acid (0.1 M), sodium citrate (0.1 M) and L-histidine (0.2 M), succinic acid (0.25 M), trehalose dihydrate (40%), sucrose (40%), hydrochloric acid (2 M) and phosphoric acid (2 M) stock solutions: Table 2 Stock solution MW (g/mol) Required concentration (mol/L) Volume (L) Required quality (9 ) Citric acid monohydrate 210.14 0.1 1.0 21.01 Sodium Citrate Dihydrate 294.1 0.1 2.0 58.82 L-histidine 155.15 0.2 1.5 46.55 Trehalose dihydrate 378.33 40% 1.5 600.00 sucrose 342.30 40% 1.0 400.00 Succinic acid 118.09 0.25 0.8 23.62 Measured by: Stock solution Initial concentration (M) Final concentration (M) Final volume (mL) Concentrated acid (mL) Water (mL) hydrochloric acid 12.1 2.0 500 82.6 417.4 Phosphoric acid 14.8 2.0 500 67.6 432.4

Weigh out the reagents according to the above table. Add an appropriate volume of Milli-Q water to dissolve the reagents. The solution was filtered through a 0.22 μm filter. Preparation of formulation buffer for dialysis

According to Table 3 below, prepare 1.0 L of each formulation for dialysis and placebo dispensing: Table 3 Formulation number pH 0.1 M citric acid monohydrate (mL) 0.1 M sodium citrate dihydrate (mL) 0.2 M L- histidine (mL) 40% trehalose dihydrate (mL) 40% sucrose (mL) Water (mL) Use the following for pH adjustment Total volume (mL) F1 5.2 61 139 137 5 662.5 1000 F2 5.7 37 163 1375 662.5 1000 F3 5.5 100 137.5 762.5 2M HCI 1000 F4 6.0 100 137.5 762.5 2M HCI 1000 F5 6.5 100 1375 762.5 2M HCI 1000 F6 5.2 61 139 125 675 1000 F7 5.7 37 163 125 675 1000 F8 5.5 100 125 775 2M HCI 1000 F9 6.0 100 125 775 2M HCI 1000 F10 6.5 100 125 775 2M HCI 1000 F11 5.5 100 137 5 762.5 2M phosphoric acid 1000 F12 6.0 100 137 5 762.5 2M phosphoric acid 1000 F13 5.5 100 137 5 762.5 0 25M Succinic acid 1000 F14 6.0 100 1375 762.5 3 25M Succinic acid 1000

Adjust the pH to the target pH ± 0.1 with an appropriate acid. Store the buffer at 4°C until use. Formulation preparation

Thaw 4 test tubes each containing 50 ml AGS-22M6E (batch number AGS22M6-VCE-02) in a room temperature water bath, and then combine them in a 250 ml bottle. For each formulation, 11 ml was dispensed and added to the dialysis cassette. Place the cassette in a beaker containing ~40-fold excess of formulation buffer and stir overnight at 2-8°C. The buffer was discarded and fresh buffer was added and stirred at 2-8°C overnight. Remove the material from the cassette and transfer to a 50 ml test tube, determine the concentration and adjust the volume with the corresponding formulation buffer so that the final concentration is 10 mg/mL. The placebo is the corresponding buffer used to formulate the product. Dispensing and stoppering

Sterile filtration and filling in Baker SG600 laminar flow hood. Use aseptic technique (Millipore Millex-GV 0.22 µm PVDF syringe filter, #SLGV033RS) to sterile filter the formulation and placebo. The sterile stoppered vial (Hollister-Stier 2-ml sterile stoppered vial, #7505ZA) was decrimped in the cover, and the stopper was removed using aseptic technique. Fill the vial with 1.0 ml of the formulated product or placebo, and then stopper. Material requirements and sample map

The material requirements and sample map are as follows: Table 4 Concentration (mg/mL) condition Number of vials Fill volume Total fill volume Total protein (mg) Total ml / formulation Total mg / formulation 10 40℃ 5 1 5.0 50 10 1 freeze/thaw cycle at -70℃/25℃ 1 1 1.0 10 10 3 freeze/thaw cycles at -70℃/25℃ 1 1 1.0 10 10 10 freeze/thaw cycles at -70℃/25℃ 1 1 1.0 10 10 Tween 20 analysis 1 1 1.0 10 10 RT shake for 24 hours 1 1 1.0 10 10 100 Number of protein concentrations tested Number of buffers for testing Total number of vials Total filling volume (mL) Protein in vial (mg) Required protein (mg) 1 14 140 140.0 1400 1680 Total number of samples: 112 condition Number of days under storage conditions 0 3 7 14 40℃ X X X X 1 freeze/thaw cycle under -70＜C/25＜C X -70＜C/25＜C under 3 freeze/thaw cycles X 10 freeze/thaw cycles under -70＜C/25＜C X RT shake for 24 hours X Time point and analysis

The time points and analysis are shown in Table 5. Table 5 Analytical analysis T=0 T=3d T=7d T=14d At -70 ℃ / 25 ℃ 1 times freeze / thaw At -70 ℃ / 25 ℃ 3 times freeze / thaw Freeze / thaw 10 times at -70 ℃ /25 ℃ RT shake for 24 hours 13-Apr-10 16-Apr-10 20-Apr-10 4/272010 pH X X Weight molar osmolality X X Visual appearance X X X X X X X X A280 X X X X X X X X Turbidity (A330) X X X X X X X X Non-reduced SDS-PAGE X X X X X X X X Reduced SDS-PAGE X X X X X X X X SE-HPLC X X X X X X X X RP-HPLC X X Performance and CIEF * Provide samples for testing 40 ℃ stability test design of liquid formulation

Place the vial of the formulation and placebo vertically in an incubator set at 40°C. At each time point, according to the sample profile, one active vial and one placebo vial of each formulation were removed from the storage conditions. The samples were frozen at -70°C and analyzed in batches at the end of the experiment. Before analysis, the samples were thawed at RT. After filtering through a 0.22 μm filter, 3 aliquots of each sample (for each sample, 70 μL aliquots) of the multiple groups were frozen at -70°C. After analytical testing, store any residual material at 2-8°C overnight in case retesting is required. After completing all analyses, the residual material was then stored at -70°C. Two frozen aliquots were used for cIEF and performance analysis. Freeze-thaw (-70 ℃ ) stability test design

Place a vial (filled with 1.0 mL) of each formulation upright in a refrigerator at -70°C for at least 4 hours, allowing freezing. For thawing, each vial was removed from the reservoir and thawed at room temperature until ice was no longer observed, and then the vial was swirled gently. This constitutes a complete freeze-thaw cycle. For each test formulation sample vial, complete one, three, and ten freeze-thaw cycles. After the final freeze-thaw cycle, all samples are evaluated by analytical testing. Then, 3 aliquots (for each sample, 70 μL aliquots) of each sample in the multiple groups were frozen at -70°C. After analytical testing, store any residual material at 2-8°C overnight in case retesting is required. After completing all analyses, the residual material was stored at -70°C. Two frozen aliquots were used for cIEF and performance analysis. Stirring test design

Fix one vial of each preparation vertically in a standard freezer box. Next, the cassette was attached to an IKA-VIBRAMAX-VXR rotary shaker set at 500 rpm at room temperature for 24 hours. The samples were then removed and stored at -70°C until analysis. Formulation standard

Take 1.2 mL of AGS-22M6E (batch number AGS22M6-VCE-02) starting material (12.5 mg/mL in 20 mM histidine pH 6.0 buffer containing 5% sucrose and 0.02% polysorbate-20) , And divided into 200 μl/vial aliquots, and then stored at -70 ℃ as the preparation standard for this test.

Visual appearance, A280 (protein concentration and drug load), A330 (turbidity), SE-HPLC, non-reducing and reducing SDS-PAGE, RP-HPLC-NPI.iCIEF and performance are used to evaluate the stability of AGS-22M6E

Visual appearance : During the test, all samples showed no color, no turbidity and no particles. Even after shaking, no particles were seen.

A280 ( protein concentration ) analysis : The results of the A280 analysis are shown in Table 6 below. Table 6 sample A280 Number of days under 40℃ 0 3 7 14 F1 0.6549 0.6274 0.6805 0.6449 F2 0.6818 0.7017 0.6929 0.6853 F3 0.7069 0.6491 0.7130 0.6990 F4 0.7056 0.7147 0.7106 0.7217 F5 0.7123 0.6837 0.7138 0.7201 F6 0.6616 0.6789 0.6935 0.6830 F7 0.6594 0.6558 0.6672 0.6730 F8 0.6986 0.6903 0.7037 0.7017 F9 0.6834 0.7002 0.7063 0.7061 F10 0.6888 0.6839 0.6937 0.7023 F11 0.7032 0.7109 0.7074 0.6950 F12 0.7040 0.6622 0.7088 0.7255 F13 0.6736 0.6754 0.6874 0.6818 F14 0.6944 0.6745 0.6878 0.7003 sample A280 0 1 freeze-thaw 3 freeze-thaw 10 freeze-thaw cycles 24 hours shaking F1 0.6549 0.6582 0.6951 0.65725 0.66017 F2 0.6818 0.6809 0.6761 0.68321 0.67565 F3 0.7069 0.6963 0.6958 0.69771 0.70847 F4 0.7056 0.7048 0.6817 0.69875 0.68629 F5 0.7123 0.7027 0.6961 0.70278 0.71533 F6 0.6616 0.6651 0.6715 0.68515 0.66747 F7 0.6594 0.6622 0.6585 0.63399 0.65046 F8 0.6986 0.6969 0.7042 0.69579 0.69878 F9 0.6834 0.6893 0.6876 0.67558 0.69247 F10 0.6888 0.6862 0.6921 0.68071 0.68312 F11 0.7032 0.6967 0.6905 0.68287 0.70169 F12 0.7040 0.6892 0.7064 0.69924 0.68928 F13 0.6736 0.6746 0.6745 0.65992 0.66715 F14 0.6944 0.6831 0.7003 0.67608 0.68892 sample Concentration (mg/mL) Number of days under 40℃ 0 3 7 14 F1 9.01 8.63 9.36 8.87 F2 9.38 9.65 9.53 9.43 F3 9.72 8.93 9.81 9.62 F4 9.71 9.83 9.77 9.93 F5 9.80 9.40 9.82 9.90 F6 9.10 9.34 9.54 9.40 F7 9.07 9.02 9.18 9.26 F8 9.61 9.50 9.68 9.65 F9 9.40 9.63 9.71 9.71 F10 9.47 9.41 9.54 9.66 F11 9.67 9.78 9.73 9.56 F12 9.68 9.11 9.75 9.98 F13 9.27 9.29 9.46 9.38 F14 9.55 9.28 9.46 9.63 sample Concentration (mg/mL) 0 1 freeze-thaw 3 freeze-thaw 10 freeze-thaw cycles 24 hours shaking F1 9.01 9.05 9.56 9.04 9.08 F2 9.38 9.37 9.30 9.40 9.29 F3 9.72 9.58 9.57 9.60 9.75 F4 9.71 9.69 9.38 9.61 9.44 F5 9.80 9.67 9.57 9.67 9.84 F6 9.10 9.15 9.24 9.42 9.18 F7 9.07 9.11 9.06 8.72 8.95 F8 9.61 9.59 9.69 9.57 9.61 F9 9.40 9.48 9.46 9.29 9.53 F10 9.47 9.44 9.52 9.36 9.40 F11 9.67 9.58 9.50 9.39 9.65 F12 9.68 9.48 9.72 9.62 9.48 F13 9.27 9.28 9.28 9.08 9.18 F14 9.55 9.40 9.63 9.30 9.48

As shown, no change in protein concentration was observed.

A330 ( turbidity ) analysis : The results of the A330 analysis are shown in Table 7 below. Table 7 sample A330 Number of days under 40℃ 0 3 7 14 Placebo Active matter Active matter Active matter Placebo Active matter F1 -0.0006 0.0591 0.1030 0.1053 0.0131 0.1223 0.1070 F2 -0.0040 0.0727 0.0934 0.1067 0.0363 F3 -0.0063 0.0585 0.0632 0.0715 0.0018 0.0736 F4 -0.0036 0.0601 0.0667 0.0730 0.0053 0.0757 F5 0.0027 0.0659 0.0720 0.0917 0.0030 0.0823 F6 0.0028 0.1018 0.0970 0.1196 0.0004 0.1269 0.1031 F7 0.0029 0.0705 0.0885 0.0925 0.0073 F8 0.0019 0.0620 0.0598 0.0801 0.0112 0.0860 F9 0.0041 0.0681 0.0776 0.0982 0.0193 0.1046 F10 0.0013 0.0628 0.0760 0.0905 0.0156 0.0886 F11 0.0036 0.0773 0.0649 0.0722 0.0100 0.0782 F12 0.0014 0.0652 0.0641 0.0861 0.0131 0.0796 F13 0.0142 0.0655 0.0660 0.0708 0.0121 0.0936 F14 0.0112 0.0659 0.0637 0.0701 0.0122 0.0857 sample A330 1 freeze-thaw 3 freeze-thaw 10 freeze-thaw cycles 24 hours shaking Active matter Active matter Placebo Active matter Placebo Active matter F1 0.0638 0.0787 0.0042 0.0755 0.0083 0.0698 F2 0.0683 0.0757 0.0075 0.0774 0.0089 0.0719 F3 0.0631 0.0645 0.0008 0.0620 0.0235 0.0669 F4 0.0593 0.0908 0.0005 0.0600 0.0036 0.0577 F5 0.0647 0.0598 0.0049 0.0685 0.0099 0.0615 F6 0.0805 0.0796 -0.0001 0.0728 0.0180 0.0737 F7 0.0714 0.0777 0.0025 0.0745 0.0100 0.0695 F8 0.0630 0.0750 0.0006 0.0696 0.0053 0.0568 F9 0.0670 0.0737 0.0034 0.0715 0.0022 0.0692 F10 0.0595 0.0750 0.0007 0.0752 0.0050 0.0676 F11 0.0559 0.0763 0.0016 0.0694 0.0013 0.0629 F12 0.0625 0.0679 0.0006 0.0695 0.0028 0.0695 F13 0.0616 0.0701 0.0028 0.0820 -0.0009 0.0759 F14 0.0611 0.0745 -0.0017 0.1033 0.0108 0.0972

As shown, formulations F1 and F6 exhibited the most significant increase in turbidity over time. At T=0, it is noted that formulation F6 has a higher turbidity, which is not observed in other formulations.

SDS-PAGE analysis : The results of SDS-PAGE analysis are shown in Figures 1A, 1B, 1C and 1D. After 14 days, by reducing SDS-PAGE, tiny low molecular weight (LMW) bands (~35kD) were observed in F1 and F6. In non-reducing SDS-PAGE analysis, F1, F2, F6 and F7 also showed tiny high molecular weight (HMW) bands (~200kD) that did not exist at T=0.

RP-HPLC analysis : Table 8 and Figure 1E show the results of RP-HPLC analysis. At t=0, the free SGD1010 (microlysis of drug MMAE) was not detected by RP-HPLC. However, after 14 days at 40℃, SGD1010 (in the range of 0.17-1.59 μM) ) And the histidine formulation is slightly faster than the citrate formulation at higher pH, where histidine/succinic acid is slightly better than histidine/phosphoric acid and histidine/HCl. Table 8 Formulation number Buffer pH Trehalose dihydrate (%) Sucrose (%) area µM SGD1010 F1 20 mM sodium citrate/citric acid 5.2 5.5 0 0 0.00 28 0.17 F2 5.7 0 0.00 31 0.18 F3 20 mM histidine/HCl 5.5 0 0.00 101 0.60 F4 6 0 0.00 127 0.76 F5 6.5 0 0.00 136 0.81 F6 20 mM sodium citrate/citric acid 5.2 0 5 0 0.00 30 0.18 F7 5.7 0 0.00 34 0.20 F8 20 mM histidine/HCl 5.5 0 0.00 96 0.57 F9 6 0 0.00 267 1.59 F10 6.5 0 0.00 124 0.74 F11 20 mM histidine/phosphoric acid 5.5 5.5 0 0 0.00 111 0.66 F12 6 0 0.00 135 0.80 F13 20 mM histidine/succinic acid 5.5 0 0.00 89 0.53 F14 6 0 0.00 106 0.63

SE-HPLC analysis : As shown in the following Table 9 and Figures 1F, 1G and 1H, at pH 5.2-5.7, for all formulations, by SE-HPLC, the level of HMW aggregates increased significantly, where the corresponding The citrate formulation showed more aggregates than the histidine formulation at pH. At similar pH, the citrate formulation showed more aggregates than the histidine formulation. The histidine formulations at pH 6.0 showed better stability than their formulations at pH 5.5 and at pH 6.5. No difference was observed between trehalose and sucrose. Table 9 % Of total points area Integral area Formulation Number of days under 40℃ Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total 1 0 19.5 1.8 96.5 1.7 83 4500 79 4661 3 19.5 36.8 61.9 1.3 1812 3050 63 4924 7 19.6 40.1 58.3 1.5 1953 2839 73 4866 14 19.6 43.1 55.5 1.4 2095 2696 68 4858 2 0 19.5 1.8 96.6 16 93 4936 83 5112 3 19.6 18.5 79.9 16 889 3832 77 4797 7 19.6 24.3 74.0 1.7 1299 3949 90 5338 14 19 6 29.4 68.6 1.9 1442 3363 95 4901 3 0 19.5 1.6 96.7 1.7 82 5078 89 5249 3 19.5 5.0 90.8 4.2 253 4603 211 5067 7 19.5 5.5 91.0 3.5 268 4463 171 4903 14 19.5 6.5 91.0 2.5 311 4333 120 4764 4 0 19 5 1.7 96.5 1.8 81 4636 85 4802 3 19.5 3.1 95.1 1.8 149 4571 87 4808 7 19.5 3.9 93.9 2.2 201 4797 110 5108 14 19 5 6.3 90.5 3.2 314 4536 162 5012 5 0 19.5 1.6 96.8 1.6 85 4991 82 5158 3 19 5 3 5 94.6 1.9 167 4534 92 4793 7 19.5 5.5 90.5 4.0 282 4622 206 5109 14 19.5 6.3 91.4 2.3 308 4430 110 4847 % Of total points area Integral area Formulation Number of days under 40℃ Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total 6 0 19.5 2.2 95.8 2.0 97 4242 88 4427 3 19 6 35.6 63.0 1.4 1754 3100 68 4921 7 19.6 38.9 59.5 1.6 1908 2915 78 4902 14 19.6 41.6 56.5 1.9 2047 2783 96 4926 7 0 19 5 1.8 96.7 1.6 89 4867 78 5034 3 19.6 18.6 79.8 17 906 3895 81 4882 7 19.6 24.2 74.2 1.7 1145 3515 78 4739 14 19.6 28.5 69.6 1.9 1360 3318 90 4768 8 0 19.5 16 96.4 2 0 82 4968 104 5154 3 19.5 3.9 94.1 1.9 184 4421 91 4696 7 19.5 5.4 92.2 2.4 259 4388 113 4760 14 19.5 6.7 90.8 2.5 322 4343 119 4785 9 0 19.6 1.2 96.5 2.3 60 4987 121 5168 3 19.5 3.3 94.6 2.1 158 4504 98 4760 7 19.5 4.2 93.6 2.3 209 4659 113 4981 14 19.5 5 2 92.1 2 7 249 4408 130 4787 10 0 19 5 16 96.7 1.7 82 4914 85 5081 3 19.5 3.4 94.8 1.9 155 4387 87 4629 7 19.5 4.8 92.9 2.3 229 4466 111 4807 14 19.5 5.9 91.5 26 276 4312 122 4710 % Of total points area Integral area Formulation Number of days under 40℃ Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total 11 0 19.5 1.6 96.8 1.6 82 4989 83 5154 3 19.5 3.6 94.4 2.0 168 4415 93 4676 7 19.5 5.3 92.5 2.2 250 4400 106 4756 14 19.6 12.9 84.8 2.3 625 4107 111 4843 12 0 19.6 1.7 96.4 1.8 97 5340 101 5538 3 19.5 3.3 94.7 1.9 159 4504 92 4755 7 19.5 4.3 93.6 2.1 214 4637 104 4955 14 19.5 6.3 89.7 4.0 319 4533 204 5060 13 0 19.5 1.9 96.7 1.7 93 4806 84 4969 3 19.5 7.1 91.0 1.9 326 4184 86 4596 7 19.5 9.8 88.1 2.1 456 4083 97 4636 14 19.5 12.6 85.0 2.4 598 4041 114 4753 14 0 19.5 1.6 96.6 1.7 82 4834 86 5002 3 19.5 4.5 93.6 1.9 204 4265 88 4557 7 19.5 5.7 92.2 2.2 267 4342 102 4711 14 19.5 6.9 89.7 3.3 349 4503 167 5018

After shaking at room temperature for 24 hours or after one, three, and ten freeze-thaw cycles, no significant changes were observed between any formulations, such as by A330, SDS-PAGE and SE-HPLC (data here Not shown) confirmed. This provides a guarantee that test samples of formulations can be taken at different time points and stored at -70°C.

Based on the results obtained from this experiment, among the 14 tested formulations, formulations F4, F9, and F14 were selected as the best, and therefore selected as the 3 formulations to be further evaluated in subsequent experiments. 6.2 Example 2- Freeze-thaw and shaking test of the bulk drug substance (BDS)

Formulations F4, F9 and F14 were prepared as described in section 6.1 above. Each of formulations F4, F9, and F14 was subjected to 1, 3, and 10 cycles of freezing at both -20°C and -70°C followed by thawing between 20°C and 25°C. Analyze the samples by visual inspection, concentration (A280) measurement, turbidity (A330) measurement, SE-HPLC, SDS-PAGE (R&NR). For 10 freeze-thaw cycle tests, samples were also analyzed by RP-HPLC NPI.

The material requirements and sample map are shown in Table 10 below: Table 10 Concentration (mg/mL) condition Number of vials Fill volume Total fill volume Total protein (mg) 10 T=0 1 3 5 3.5 35 10 1 freeze/thaw cycle at -20℃/25℃ 1 1 1.0 10 10 3 freeze/thaw cycles at -20℃/25℃ 1 1 1.0 10 10 10 freeze/thaw cycles at -20℃/25℃ 1 1 1.0 10 10 1 freeze/thaw cycle at -70℃/25℃ 1 1 1.0 10 10 3 freeze/thaw cycles at -70℃/25℃ 1 1 1.0 10 10 10 freeze/thaw cycles at -70℃/25℃ 1 1 1.0 10 10 Shake for 24 hours at RT 1 3.5 3.5 35 Number of protein concentrations tested Number of buffers for testing Total number of vials Total filling volume (mL) Protein in vial (mg) Required protein (mg) 1 3 twenty four 39 390 468 Note: • Vial: 5 mL sterile screw cap polycarbonate bottle (Nalgene 5-ml, #3500-05) • BDS freeze/thaw: 1 mL is filled in a 5 mL polycarbonate bottle. • BDS shaking: 3.5 mL is filled in a 5 mL polycarbonate bottle.

Table 11 below lists the analysis and time points. Table 11 Analytical analysis T=0 1 freeze/thaw at -70℃/25℃ Freeze/thaw 3 times at -70℃/25℃ Freeze/thaw 10 times at -70℃/25℃ 1 freeze/thaw at -20℃/25℃ Freeze/thaw 3 times at -20℃/25℃ Freeze/thaw 10 times at 20℃/25℃ RT shake for 24 hours pH X Weight molar osmolality X Visual appearance X X X X X X X X A280 X X X X X X X X Turbidity (A330) X X X X X X X X Non-reducing SDS-PAGE X X X X X X X X Reductive SDS-PAGE X X X X X X X X SE-HPLC X X X X X X X X RP-HPLC-NPI X X X X X X X X HIAC X X Performance and CIEF Provide samples for testing

A 24 hour agitation test at RT was also performed for each formulation, and the visual, concentration (A280), turbidity (A330), SE-HPLC and HIAC of all test samples were analyzed. The selected samples from the above tests are also used for iCIEF and efficacy tests.

The preparations are packed and stoppered, the agitation test design, the freeze-thaw test design and the preparation standards are described below. Dispensing and stoppering

Sterile filtration and filling in Baker SG600 laminar flow hood. Use aseptic technique (Millipore Millex-GV 0.22 µm PVDF syringe filter, #SLGV033RS) to sterile filter the formulation and placebo. Using a sterile tip for a 5 mL electronic pipette, transfer the filtered AGS-22M6E and the filtered formulation buffer (placebo) to a sterile screw cap polycarbonate bottle (Nalgene 5-ml, #3500-05). Stirring test design

Fix a vial/mixture vertically in a standard freezer box. Next, the cassette was attached to an IKA-VIBRAMAX-VXR rotary shaker set at 500 rpm at room temperature for 24 hours. The samples were then removed and stored at -70°C until analysis. After filtering through a 0.22 μm filter, 3 aliquots of each sample (for each sample, 70 μL aliquots) of the multiple groups were frozen at -70°C. After analytical testing, store any residual material at 2-8°C overnight in case retesting is required. After completing all analyses, the residual material was then stored at -70°C. Two frozen aliquots were used for cIEF and performance analysis. Freeze-thaw (-70 ℃ and -20 ℃ ) stability test design

Place 1 vial/formulation (1 ml filled in a 5 mL polycarbonate bottle) upright in the refrigerator at -70°C and -20°C for at least 4 hours, allowing freezing. For thawing, each vial was removed from the reservoir and thawed at room temperature (20-25°C) until ice was no longer observed, then the vial was swirled gently. This constitutes a complete freeze-thaw cycle. For each test formulation sample vial, ten freeze-thaw cycles are completed. After the final freeze-thaw cycle, all samples are evaluated by analytical testing. The samples were analyzed by the following methods: visual appearance, A280/A248, turbidity (A330), SE-HPLC, RP-HPLC-NPI, and SDS-PAGE (R & NR). After filtering through a 0.22 μm filter, 3 aliquots of each sample (for each sample, 70 μL aliquots) of the multiple groups were frozen at -70°C. After analytical testing, store any residual material at 2-8°C overnight in case retesting is required. After completing all analyses, the residual material was then stored at -70°C. Two frozen aliquots were used for cIEF and performance analysis. Formulation standard

Take 15 mL of 12.8 mg/mL 5.0% sucrose containing AGS-22M6E starting material, 0.02% Tween 20 pH 6.0, and aliquot 500 μl/vial, and then store at -70℃ as the preparation for this test物standards. result

Visual appearance : The visual appearance of all samples was analyzed in this test, and no particles were seen, even after shaking.

A280 and A330 analysis : The A280 and A330 data of the formulations subjected to different conditions in this test are summarized in Table 12 below. As shown, there was no change in protein concentration under either shaking or freezing and thawing conditions. In addition, the turbidity did not increase after freezing and thawing or shaking. Table 12 Point in time Formulation Dilution factor A330 A280 A280 (Placebo A280 has been subtracted) Concentration (mg/mL) A330 undiluted T=0, BDS 4 9 14 20 20 20 0.012 0.021 0.004 0.741 0.811 0.820 0.728 0.795 0.789 10.01 10.94 10.86 0.083 0.091 0.103 oscillation 4 9 14 20 20 20 0.011 0.027 0.001 0.749 0.827 0.820 0.736 0.811 0.789 10.12 11.16 10.86 0.089 0.960 0.107 1 time -20℃ freeze-thaw 4 9 14 20 20 20 0.006 0.026 0.005 0.765 0.812 0.823 0.752 0.796 0.792 10.34 10.96 10.90 0.097 0.098 0.112 3 times -20℃ freeze-thaw 4 9 14 20 20 20 0.003 0.030 0.003 0.779 0.819 0.821 0.766 0.803 0.790 10.54 11.05 10.87 0.096 0.098 0.108 10 times -20℃ freeze-thaw 4 9 14 20 20 20 0.001 0.031 0.008 0.783 0.822 0.818 0.770 0.806 0.787 10.59 11.08 10.83 0.097 0.109 0.128 Freeze-thaw at -70℃ once 4 9 14 20 20 20 0.002 0.028 0.001 0.780 0.839 0.816 0.767 0.823 0.785 10.55 11.32 10.80 0.114 0.120 0.108 3 times -70℃ freeze-thaw 4 9 14 20 20 20 0.004 0.031 0.003 0.789 0.846 0.830 0.776 0.830 0.799 10.67 11.42 11.00 0.095 0.097 0.107 10 times -70℃ freeze-thaw 4 9 14 20 20 20 0.006 0.031 0.002 0.797 0.833 0.827 0.784 0.817 0.796 10.78 11.24 10.95 0.091 0.090 0.093

SDS-PAGE analysis : SDS-PAGE analysis results are shown in Figures 2A and 2B. As shown, for the shaking test sample and for the freeze-thaw sample, no change was seen by SDS-PAGE. Both the reducing and non-reducing gels are comparable to the formulation standards.

RP-HPLC analysis : For 10 freeze-thaw cycle samples, perform RP-HPLC analysis. In any formulation, no sign of the SGD1010 peak was seen, as analyzed by RP-HPLC (data not shown here).

SE-HPLC analysis : The results of the SE-HPLC analysis are summarized in Table 13 below. As shown, for any of the three formulations (F4, F9, and 14) or placebo, no differences were observed between TO and shake samples or freeze-thaw samples. Table 13 Sample name condition % Of total points area Integral area Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total F4, BDS T0 19.6 1.3 95.7 3.0 66 4955 154 5175 RT shake for 24 hours 19.5 1.2 96.2 2.6 64 4937 134 5134 1 F/T -20℃ 19.6 1.2 95.9 2.9 63 4913 150 5125 3 F/T -20℃ 19.5 1.2 96.1 2.7 63 4960 138 5161 10 F/T -20℃ 19.5 1.2 96.3 2.4 64 4935 125 5123 1 F/T -70℃ 19.6 1.2 96.0 2.7 63 4929 141 5132 3 F/T -70℃ 19.5 1.2 96.2 2.5 64 5006 132 5202 10 F/T -70℃ 19.6 1.3 95.9 2.8 68 4981 143 5192 F9, BDS T0 19.6 1.3 95.6 3.1 66 5002 164 5232 RT shake for 24 hours 19.6 1.3 96.4 2.3 65 4980 120 5164 1 F/T -20℃ 19.5 1.3 95.8 2.9 71 5007 152 5229 3 F/T -20℃ 19.5 1.2 96.5 2.3 63 4997 119 5179 10 F/T -20℃ 19.6 1.2 96.1 2.7 64 4944 137 5145 1 F/T -70℃ 19.6 1.3 96.3 2.4 64 4918 125 5107 3 F/T -70℃ 19.5 1.2 96.3 2.5 63 4978 127 5168 10 F/T -70℃ 19.5 1.2 96.1 2.6 65 4996 136 5197 F14, BDS T0 19.6 1.3 95.7 3.0 72 5190 161 5423 RT shake for 24 hours 19.6 1.3 96.1 2.6 67 5052 138 5256 1 F/T -20℃ 19.6 1.4 95.7 2.9 74 5090 154 5319 3 F/T -20℃ 19.5 1.2 96.1 2.7 65 5047 142 5254 10 F/T -20℃ 19.6 1.3 96.3 2.4 66 5028 127 5221 1 F/T -70℃ 19.6 1.2 96.4 2.4 66 5106 125 5297 3 F/T -70℃ 19.5 1.2 96.3 2.4 65 5075 129 5269 10 F/T -70℃ 19.6 1.3 95.5 3.2 71 5096 169 5336

Analyze the SEC curves obtained for each BDS formulation under each test condition (data not shown here). Compared with the BDS at T=0, no difference was noticed in any formulation under any conditions.

Table 14 below summarizes the HIAC data of the BDS samples for each of the 3 formulations tested. A comparison of the results of samples before and after shaking at RT for 24 hours showed that less than 100 particles were in the range between 10 µm and 25 µm, and for all formulations, less than 2 particles were in the 25 µm range. Table 14 Average 3 rounds of total cumulative counts /mL Size (um) condition Sample name Formulation 2 5 7.5 10 15 20 25 4 47 10 7 5 5 3 2 Placebo 9 73 30 17 8 5 2 0 No oscillation 14 73 17 12 10 3 0 0 4 382 77 32 15 2 2 0 AGS22M6E 9 135 45 28 18 7 0 0 14 365 138 68 47 15 3 2 4 298 127 78 47 twenty three 3 0 Placebo 9 365 155 88 60 13 2 0 Shake for 24 hours at RT 14 407 118 47 25 5 0 0 4 368 127 62 42 18 5 2 AGS22M6E 9 557 192 108 73 20 3 2 14 967 333 165 97 32 12 2

Formulations F9 and F14 showed a slight increase in cumulative counts after agitation, and F4 showed the comparability before and after agitation, as shown in Figure 2C (all counts at 10 and 25 μm were below the USP limit).

In general, the results showed that all three tested BDS formulations exhibited excellent stability under freeze-thaw cycles and agitation treatment, and no change relative to T=0 was seen in any sample by any analysis method. 6.3 Example 3- Parallel BDS and drug (DP) formulation test

This test is carried out in conjunction with the test described in Section 6.2 above. The formulation composition and materials used in this test are the same as those described in section 6.2.

Material requirements and sample maps are shown in the table below. Table 15 Concentration (mg/mL) condition Number of vials Fill volume Total fill volume Total protein (mg) Total ml / formulation Total mg / formulation 10 BDS, at 2-8℃ 5 1 5.0 50 10 BDS, at -70℃ 6 1 6.0 60 10 DP, at 2-8℃ 8 5 40.0 400 10 DP, under 25℃/60%RH 6 5 30.0 300 10 DP, under 40℃/75%RH 6 5 30.0 300 111 1110 Number of protein concentrations tested Number of buffers for testing Total number of vials Total filling volume (mL) Protein in vial (mg) Required protein (mg) 1 3 93 333 3330 3996

The time points and analysis are described in the table below. Table 16 Analytical analysis Pre-Lyo T=0 T=2 weeks T=4 weeks T=8 weeks T=12 weeks T=0.1 1-June-10 15-June-10 29-June-10 27-July-10 24-August-10 pH X X Weight molar osmolality X X Visual appearance (before restoration) X X X X X Visual appearance (BDS or after restoration) X X X X X X Recovery time (DP only) X X X X X A280 X X X X X X Turbidity (A330) X X X X X X Non-reduced SDS-PAGE X X X X X X Reductive SDS-PAGE X X X X X X SE-HPLC X X X X X X RP-HPLC-NPI X X X X X X Residual moisture (DP only) X X Performance and CIEF* Provide samples for testing Note : Freeze the samples of the liquid arm of the test at -70°C until the samples from the lyophilized group are prepared. Then, put the frozen liquid sample under the condition, and put the freeze-dried sample under the condition at the same time, so that the t=0 of the liquid group and the lyophilized group of the test are consistent.

The specific freeze-drying cycle parameters are summarized in the table below. After completion of lyophilization, the vial was vacuum stoppered at 50 mT. Table 17 Step number step Temperature or temperature change rate Time (min) Pressure ( mTorr ) 1 Load/balance 5℃ 60 2 Change from 5℃ to 0℃ 0.5℃/min 10 3 maintain 0℃ 60 4 Change from 0℃ to -45℃ 1℃/min 45 5 maintain -45℃ 840 6 Gasp -45℃ 60 50 7 Change from -45℃ to -15℃ 0.3°C/min 100 50 8 maintain -15℃ 5040 50 9 Change from -15℃ to 35℃ 0.2℃/min 250 50 10 maintain 35°C 360 50 11 Change from 35℃ to 5℃ 0.5℃/min 60 50 12 maintain Keep 5℃ 60 50 Design of stability test of liquid formulation at 2-8 ℃ and -70 ℃

Place the vial of the formulation and placebo vertically in a refrigerator set at -70°C and an incubator set at 2-8°C. At each time point, according to the sample profile, one active vial and one placebo vial of each formulation were removed from storage conditions for analytical testing. After analytical testing, store any residual material at 2-8°C in case there is a need for retesting. The aliquots were stored at -70°C and used for cIEF and activity testing. Stability test design of freeze-dried formulations at 2-8 ℃ , 25 ℃ and 40 ℃

Place the formulation and the placebo vial vertically in an incubator set at 2-8°C, an incubator set at 25°C/60%RH, and an incubator set at 40°C/75%RH. At each time point, according to the sample profile, one active vial and one placebo vial of each formulation were removed from the storage conditions for analytical testing. After analytical testing, store any residual material at 2-8°C in case there is a need for retesting. The aliquots were stored at -70°C and used for cIEF and activity testing.

The formulation standard used in this test is the same as the formulation standard in section 6.2 above.

Freeze-drying cycle analysis : A typical freeze-drying cycle includes freezing, primary drying and secondary drying steps. During the freezing and drying process, several individual indicators are mentioned, which can be followed by sublimation of ice, such as the readings of thermocouple probes placed in placebo sample vials, capacitance pressure gauges and Pirani vacuum The difference between the pirani gauge pressure readings will be consistent later, and the "dew point" measurement that tracks the relative humidity changes in the headspace of the chamber.

By comparing the average product thermocouple temperature, capacitance pressure gauge/Pirani gauge reading difference, and dew point profile, it can be confirmed that each is well correlated with others.

Stability of BDS formulations : at T=0, 2, 4, 8 and 12 weeks, evaluate the storage conditions at 2-8℃ and -70℃ and the storage conditions at 2-8℃, 25℃ and 40℃ The stability of the BDS formulation below. Analyze liquid and reconstitute lyophilized samples by concentration (A280), turbidity (A330), SE-HPLC, SDS-PAGE (R and NR) and RP-HPLC NPI at each time point. For lyophilized drugs (DP), measure the weight molar osmolality at only t=0 before lyophilization and after recovery; measure the appearance of the cake and the recovery time at each time point; and when only T=0 and T= 12 weeks, measure Karl Fischer (residual moisture).

Visual appearance and recovery time : The cakes of all three formulations are equivalent. Both the active and placebo of all three formulations formed white slightly cracked cakes with shiny surfaces. All maintain a sound structure. There is no difference between active and placebo. There are no differences between these formulations. For all formulations stored under different conditions for 2, 4, 8 and 12 weeks, the visual appearance of the cake was similar to T=0, as shown in the table below. Table 18 Formulation Protein concentration mg/ml Pie appearance Recovery time (seconds)^ Visual appearance 40℃ 25℃ 2-8°C 2 weeks 4 10 White pie, slightly cracked, shiny surface twenty three 19 twenty one Clear, colorless, no particles 9 10 17 twenty one 25 14 10 twenty one twenty two twenty two 4 Placebo twenty two 9 twenty one 14 twenty two 4 weeks 4 10 White pie, slightly cracked, shiny surface 29 25 twenty three Clear, colorless, no particles 9 10 17 twenty three twenty one 14 10 20 twenty one 27 4 Placebo 29 9 26 14 20 8 weeks 4 10 White pie, slightly cracked, shiny surface 25 28 29 Clear, colorless, no particles 9 10 25 twenty three 31 14 10 twenty four twenty two 33 4 Placebo 28 9 twenty two 14 19 12 weeks 4 10 White pie, slightly cracked, shiny surface twenty two 28 twenty four Clear, colorless, no particles 9 10 20 twenty two 27 14 10 twenty three 25 twenty three 4 Placebo 28 9 20 14 28

Moisture analysis : After completion of lyophilization, an active vial and a placebo vial from each formulation were distributed for residual moisture testing. As shown in the following table and Figure 3A, the residual moisture of the actives of F4 and F14 and the placebo are very close, in the range of 0.24 to 0.70%. F9 has higher residual moisture than F4 and F14 at each time point. Table 19 Residual moisture% SD t=0 t=12 weeks; 2-8℃ t=12 weeks; 25℃ t=12 weeks; 40℃ T=0 2-8°C 25℃ 40℃ Active matter F4 0.24 0.25 0.42 0.54 0.03 0.01 0.02 0.03 F9 0.76 0.55 0.61 0.75 0.02 0.00 0.02 0.02 F14 0.24 0.24 0.29 0.55 0.01 0.02 0.01 0.01 Placebo F4 0.29 0.28 0.56 0.70 0.01 0.02 0.02 0.01 F9 0.77 0.73 1.15 1.14 0.01 0.03 0.01 0.02 F14 0.21 0.22 0.44 0.69 0.01 0.02 0.01 0.01 The result represents the average of three determinations of 1 vial

After incubating the samples under different conditions for 12 weeks, the residual moisture of both the active and the placebo of each formulation was tested again. The residual moisture of all three formulations stored at 2-8°C for 12 weeks is similar to t=0. F4 and F14 have increased moisture relative to t=0 after being stored at 25°C and 40°C for 12 weeks.

For all time points tested, after reconstitution with 4.7 mL WFI, all reconstituted formulations and placebo were colorless and clear. No visible particles were observed. BDS samples stored under all conditions at all time points are also clear and colorless. No visible particles were observed.

A280 and weight molar osmotic concentration analysis : Before filling and freeze-drying, check the protein concentration of the formulation, repeat it twice, and find that it is within ± 1 mg/mL of the target concentration of 10 mg/mL. After reconstitution with 4.7 mL WFI, the protein concentration of the formulation was within ± 1 mg/mL of the BDS before lyophilization (see Table 20 below, BDS, t=0). Also before filling and after recovery, test the weight molar osmolality of the prepared sample and buffer, and repeat twice. The weight molar osmolality before lyophilization and after reconstitution is between 187 and 194 mOsm/kg.

The protein concentration results of BDS and DP samples stored under different conditions are shown in Table 20 below and Figure 3B. Under most conditions, there is no change in protein concentration. However, BDS samples stored at 2-8°C showed an unexpected increase in protein concentration. There may be two reasons: 1) The condensation present in the vial may not be completely incorporated after inversion. Although efforts have been made to capture all the condensation from the side of the bottle, some condensation may be trapped in the bottle cap; 2) If the bottle has a defective thread, some sample evaporation may occur, but this problem is not seen in the filling of 4 mL API In these bottles and stored at 2-8°C for 12 weeks in the previous test. Table 20 (12-week parallel BDS and DP formulation test A280 and concentration data) A280 Concentration (mg/mL) BDS- Number of weeks at -70 ℃ Formulation 0 2 4 8 12 0 2 4 8 12 4 0.757 0.737 0.735 0.740 0.727 10.4 10.1 10.1 10.2 10.0 9 0.735 0.725 0.749 0.727 0.736 10.1 10.0 10.3 10.0 10.1 14 0.743 0.740 0.746 0.764 0.742 10.2 10.2 10.3 10.5 10.2 BDS- Number of weeks at 2-8 ℃ Formulation 0 2 4 8 12 0 2 4 8 12 4 0.757 0.749 0.763 0.801 0.812 10.4 10.3 10.5 11.0 11.2 9 0.735 0.748 0.776 0.817 0.918 10.1 10.3 10.7 11.2 12.6 14 0.743 0.759 0.778 0.823 0.824 10.2 10.4 10.7 11.3 11.3 A280 Concentration (mg/mL) Freeze-dried DP- the number of weeks at 2-8 ℃ Formulation 0 0.1 2 4 8 12 0 0.1 2 4 8 12 4 0.757 0.757 0.745 0.778 0.773 0.747 10.4 10.4 10.2 10.7 10.6 10.3 9 0.735 0.769 0.743 0.777 0.782 0.769 10.1 10.6 10.2 10.7 10.8 10.6 14 0.743 0.800 0.766 0.815 0.751 0.769 10.2 11.0 10.5 11.2 10.3 10.6 Lyophilized DP- number of weeks at 25 ℃ Formulation 0 0.1 2 4 8 12 0 0.1 2 4 8 12 4 0.757 0.757 0.751 0.765 0.761 0.751 10.4 10.4 10.3 10.5 10.5 10.3 9 0.735 0.769 0.751 0.778 0.768 0.774 10.1 10.6 10.3 10.7 10.6 10.6 14 0.743 0.800 0.743 0.784 0.770 0.765 10.2 11.0 10.2 10.8 10.6 10.5 Lyophilized DP- the number of weeks at 40 ℃ Formulation 0 0.1 2 4 8 12 0 0.1 2 4 8 12 4 0.757 0.757 0.746 0.766 0.758 0.736 10.4 10.4 10.3 10.5 10.4 10.1 9 0.735 0.769 0.750 0.776 0.763 0.766 10.1 10.6 10.3 10.7 10.5 10.5 14 0.743 0.800 0.772 0.807 0.771 0.755 10.2 11.0 10.6 11.1 10.6 10.4 Table 21 (12-week parallel BDS and DP formulation test weight molar osmolality data) Formulation Reading 1 Reading 2 average value Placebo 4 193 194 194 191 T=0 , Lyo 9 197 195 196 192 14 193 194 194 186 4 189 190 190 188 T=0 , BDS 9 189 190 190 186 14 188 186 187 180

A330 analysis : The A330 measurements of both the BDS and DP reconstituted active vials and placebo vials are shown in Table 22 below and Figure 3C. The A330 value of the active vial was slightly higher than that of the placebo, indicating that the AGS-22M6E protein promoted turbidity of the formulation. There was no significant increase in the turbidity of both the active and placebo samples stored under all conditions. Table 22 BDS- Number of weeks at -70 ℃ BDS- Number of weeks at 2-8 ℃ Formulation 0 2 4 8 12 Formulation 0 2 4 8 12 4 0.083 0.085 0.084 0.101 0.091 4 0.083 0.089 0.082 0.104 0.111 9 0.091 0.09 0.085 0.096 0.086 9 0.091 0.09 0.091 0.103 0.114 14 0.103 0.094 0.097 0.104 0.103 14 0.103 0.099 0.095 0.111 0.112 4 placebo 0.009 4 placebo 0.009 0.003 0.004 0.017 0.008 9 placebo 0.01 9 placebo 0.01 0.009 0.005 0.018 0.015 14 placebo 0.01 14 placebo 0.01 0.005 0.002 0.022 0.011 Freeze-dried DP- the number of weeks at 2-8 ℃ Lyophilized DP- the number of weeks at 25 ℃ Formulation 0 0.1 2 4 8 12 Formulation 0 0.1 2 4 8 12 4 0.083 0.104 0.109 0.107 0.128 0.124 4 0.083 0.104 0.108 0.103 0.107 0.12 9 0.091 0.104 0.105 0.104 0.111 0.117 9 0.091 0.104 0.111 0.104 0.105 0.116 14 0.103 0.114 0.12 0.116 0.13 0.12 14 0.103 0.114 0.117 0.105 0.124 0.119 4 placebo 0.009 0.009 4 placebo 0.009 0.009 9 placebo 0.01 0.03 9 placebo 0.01 0.03 14 placebo 0.01 0.02 14 placebo 0.01 0.02 Lyophilized DP- the number of weeks at 40 ℃ 0 week is before lyophilization (BDS t=0) 0.1 week is after lyophilization (previously marked as t=0 lyophilization) Formulation 0 0.1 2 4 8 12 4 0.083 0.104 0.114 0.101 0.109 0.119 9 0.091 0.104 0.11 0.103 0.107 0.109 14 0.103 0.114 0.109 0.105 0.149 0.118 4 placebo 0.009 0.009 0.012 0.008 0.015 0.016 9 placebo 0.01 0.03 0.013 0.013 0.02 0.017 14 placebo 0.01 0.02 0.007 0.009 0.014 0.014

SDS-PAGE analysis : Figures 3D and 3E show the SDS-PAGE analysis of BDS T=0 (before freeze-drying) and T=12-week samples under storage conditions of 2-8℃ and -70℃. Figure 3F, 3G, and 3H show the SDS-PAGE analysis of DP samples at T=0 and T=12 weeks under storage conditions of 2-8℃, 25℃ and 40℃. For all formulations stored under all conditions for 12 weeks, no significant changes were seen.

RP-HPLC analysis : The RP-HPLC NPI method was also used to test all BDS and DP samples in this test. No SGD1010 peak was observed in any formulation under any conditions (data not shown here). For all the controls operated at each time point, the spike recoveries of SGD1010 in the formulation standard were about 100%. At T=4 weeks, 8 weeks, and 12 weeks, although a new dilution from the 10 mM SGD1010 stock solution was used with the freshly prepared diluent, the SGD1010 peak was split and incorporated into the standard of the formulation. SGD1010 is not separated (data not shown here). This is consistent throughout the sequence (data not shown here). The calculated recovery rate uses the combined area of the split peaks.

SE-HPLC analysis : Table 23 below summarizes the SE-HPLC data of the formulation standards operating at each time point in this test. The data shows the percentage change level of the main peak and the back peak in different rounds of the same sample. Table 23 Formulation standard % Of total points area Integral area Week number injection Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total 0 weeks 1 19.6 1.3 95.8 2.9 423 30906 924 32253 2 19.6 1.3 95.8 2.9 404 30758 934 32096 3 19.5 1.3 96.0 2.7 410 30915 876 32200 average value 19.5 1.3 95.9 2.8 412 30860 911 32183 CV% 0.0 2.2 0.1 3.5 2.4 0.3 3.4 0.2 2 weeks 1 20.7 1.2 95.7 3.1 422 32516 1047 33986 2 19.8 1.2 95.7 3.0 403 31099 986 32488 3 19.7 1.2 95.7 3.1 405 31201 1007 32613 average value 19.7 1.2 95.7 3.1 410 31606 1013 33029 CV% 0.0 0.1 0.0 1.0 2.6 2.5 3.1 2.5 4 weeks 1 19.8 1.2 95.3 3.6 379 30322 1133 31834 2 19.8 1.2 95.4 3.4 382 30449 1100 31931 3 19.8 1.2 95.5 3.3 400 30918 1066 32384 average value 19.8 1.2 95.4 3.4 387 30563 1100 32050 CV% 0.0 2.0 0.1 3.9 3.0 1.0 3.0 0.9 8 weeks 1 20.2 1.2 95.5 3.3 411 31427 1074 32911 2 20.2 1.2 95.6 3.1 407 31417 1033 32857 3 20.1 1.3 95.7 3.0 417 31657 999 33073 average value 20.2 1.2 95.6 3.1 412 31500 1035 32947 CV% 0.1 0.8 0.1 3.9 1.2 0.4 3.6 0.3 total average value 19.9 1.2 95.6 3.1 405 31132 1015 32552 stdev 0.3 0.0 0.21 0.2 13.8 585.6 76.3 596.1 CV% 1.7 2.6 0.22 7.7 3.4 1.9 7.5 1.8 % Of total points area Integral area sample Week number Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total Average value of formulation standard 0 19.5 1.3 95.9 2.8 412.1 30859.6 911.3 32183 2 19.7 1.2 95.7 3.1 409.9 31605.7 1013.4 33029 4 19.8 1.2 95.4 3.4 386.7 30562.9 1100.0 32050 8 20.2 1.2 95.6 3.1 411.6 31500.2 1035.2 32947 12 19.4 1.2 95.1 3.6 384.5 29485.9 1125.2 30996

Table 24 below summarizes the SE-HPLC data, which summarizes the percentages of HMW peak, main peak, and LMW peak of BDS samples stored at 2-8°C and -70°C for 12 weeks (data not shown here). The percentage changes of the main peak and the back peak at different time points are very similar to the changes seen in the formulation standard (data not shown here). Therefore, it can be inferred that no major changes occurred, and all three formulations were stable in liquid form at 2-8°C and -70°C for 12 weeks. No major differences were observed in the formulations. Table 24 % Of total points area Integral area Sample name Number of weeks at 2-8 ℃ Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total F4 0 19.6 1.3 95.9 2.9 412 31291 940 32642 2 19.7 1.3 95.6 3.1 445 32429 1056 33930 4 19.8 1.3 95.4 3.3 425 32200 1119 33744 8 20.2 1.3 95.9 2.8 479 35060 1032 36571 12 19.4 1.3 95.3 3.4 449 33754 1216 35419 F9 0 19.6 1.3 95.9 2.8 413 31522 932 32866 2 19.7 1.3 95.6 3.1 433 32137 1037 33607 4 19.8 1.3 95.5 3.2 438 32540 1100 34078 8 20.1 1.3 95.9 2.8 493 35546 1044 37082 12 19.4 1.3 95.4 3.3 494 36686 1285 38465 F14 0 19.6 1.3 95.8 2.9 439 32730 981 34150 2 19.7 1.3 95.6 3.1 444 31954 1021 33418 4 19.8 1.3 95.5 3.2 461 33355 1120 34936 8 20.1 1.4 95.9 2.8 503 35252 1013 36768 12 19.4 1.3 95.5 3.2 473 34069 1125 35667 % Of total points area Integral area Sample name Number of weeks at 70 ℃ Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total F4 0 19.6 1.3 95.9 2.9 412 31291 940 32642 2 19.7 1.3 95.5 3.2 434 31610 1069 33112 4 19.8 1.3 95.4 3.3 427 31179 1070 32677 8 20.1 1.3 95.9 2.8 439 31922 941 33302 12 19.4 1.3 95.4 3.3 396 29995 1041 31432 F9 0 19.6 1.3 95.9 2.8 413 31522 932 32866 2 19.7 1.3 95.6 3.1 429 31119 1019 32567 4 19.8 1.3 95.4 3.3 426 31384 1079 32889 8 20.1 1.3 95.9 2.8 442 32178 938 33559 12 19.4 1.3 95.4 3.3 397 30027 1051 31475 F14 0 19.6 1.3 95.8 2.9 439 32730 981 34150 2 19.7 1.3 95.6 3.1 431 31071 1000 32502 4 19.8 1.3 95.4 3.3 437 31571 1097 33105 8 20.1 1.3 95.9 2.8 454 32541 952 33947 12 19.4 1.3 95.4 3.3 412 30512 1046 31970

The following table 25 summarizes the SE-HPLC data, which summarizes the HMW peak, main peak and LMW peak of freeze-dried AGS-22M6E stored at 2-8°C, 25°C/60%RH and 40°C/75%RH for 12 weeks percentage. Figure 3I shows the SE-HPLC data in the curves of AGS-22M6E BDS and DP stored under different conditions. The main peak and back peak and the percentage changes at different time points are very similar to those seen in the formulation standard, indicating that there is no major change in the formulation sample. The SE-HPLC overlap of different DP formulations stored under different conditions was also analyzed (data not shown here). No difference was observed before and after lyophilization. All three formulations were stable in lyophilized form after 12 weeks at 2-8°C, 25°C and 40°C. With the exception of the acceptable stability profile seen for BDS in each case (at T=0 and after shaking and freeze-thaw tests), all formulations behaved similarly and high quality products were obtained after lyophilization. Table 25 % Of total points area Integral area Sample name Number of weeks at 2-8 ℃ Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total F4 0 19.6 1.3 95.9 2.9 412 31291 940 32642 0.1 19.6 1.2 95.8 2.9 418 32092 972 33483 2 19.7 1.2 95.7 3.1 400 31564 1027 32991 4 19.8 1.2 95.6 3.2 413 33168 1106 34687 8 20.1 1.3 95.8 2.9 449 32603 997 34049 12 19.4 1.3 95.2 3.5 403 30358 1124 31885 F9 0 19.6 1.3 95.9 2.8 413 31522 932 32866 0.1 19.6 1.2 95.9 2.9 418 32453 972 33842 2 19.8 1.3 95.5 3.2 432 31657 1068 33157 4 19.8 1.3 95.4 3.3 441 32915 1145 34502 8 20.1 1.3 95.8 2.9 459 33035 1000 34494 12 19.4 1.3 95.2 3.5 411 31112 1149 32672 F14 0 19.6 1.3 95.8 2.9 439 32730 981 34150 0.1 19.6 1.3 95.8 2.9 450 33238 1005 34693 2 19.8 1.3 95.4 3.2 461 32915 1110 34486 4 19.8 1.3 95.4 3.3 472 34323 1178 35972 8 20.1 1.3 95.8 2.9 468 33414 999 34881 12 19.4 1.3 95.3 3.4 424 31239 1108 32771 % Of total points area Integral area Sample name Number of weeks at 25 ℃ Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total F4 0 19.6 1.3 95.9 2.9 412 31291 940 32642 0.1 19.6 1.2 95.8 2.9 418 32092 972 33483 2 19.8 1.2 95.6 3.2 406 31761 1069 33236 4 19.8 1.2 95.4 3.3 410 32451 1138 33999 8 20.2 1.3 95.7 3.0 431 32611 1019 34061 12 19.4 1.3 95.2 3.5 420 30805 1142 32367 F9 0 19.6 1.3 95.9 2.8 413 31522 932 32866 0.1 19.6 1.2 95.9 2.9 418 32453 972 33842 2 19.7 1.3 95.4 3.3 438 31863 1089 33389 4 19.8 1.3 95.4 3.4 440 32810 1155 34405 8 20.2 1.3 95.7 3.0 454 32933 1015 34402 12 19.4 1.3 95.2 3.5 417 31201 1153 32770 F14 0 19.6 1.3 95.8 2.9 439 32730 981 34150 0.1 19.6 1.3 95.8 2.9 450 33238 1005 34693 2 19.7 1.3 95.4 3.2 456 32561 1104 34121 4 19.8 1.3 95.3 3.3 470 33734 1180 35383 8 20.1 1.4 95.7 3.0 480 33192 1027 34698 12 19.4 1.3 95.2 3.4 445 31605 1142 33192 % Of total points area Integral area Sample name Number of weeks at 40 ℃ Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total F4 0 19.6 1.3 95.9 2.9 412 31291 940 32642 0.1 19.6 1.2 95.8 2.9 418 32092 972 33483 2 19.8 1.3 95.4 3.4 414 31498 1120 33032 4 19.8 1.3 95.4 3.3 448 32735 1144 34327 8 20.2 1.4 95.5 3.1 455 32159 1045 33659 12 19.4 1.4 95.1 3.5 450 30523 1137 32109 F9 0 19.6 1.3 95.9 2.8 413 31522 932 32866 0.1 19.6 1.2 95.9 2.9 418 32453 972 33842 2 19.8 1.3 95.4 3.3 428 32091 1103 33622 4 19.8 1.2 95.6 3.2 414 32985 1120 34518 8 20.2 1.3 95.6 3.1 426 32104 1039 33569 12 19.4 1.3 95.1 3.6 422 30813 1158 32393 F14 0 19.6 1.3 95.8 2.9 439 32730 981 34150 0.1 19.6 1.3 95.8 2.9 450 33238 1005 34693 2 19.7 1.3 95.5 3.2 436 32383 1099 33918 4 19.8 1.3 95.5 3.2 450 33290 1126 34866 8 20.2 1.5 95.5 3.0 507 32935 1044 34486 12 19.4 1.5 95.1 3.5 475 30996 1132 32604 6.4 Example 4- Development of Freeze - Drying Cycle

The formulation F4 described in the above section was selected for this further development experiment of freeze-drying cycle. The freeze-drying cycle parameters are shown in the table below. After completion of lyophilization, the vial was vacuum stoppered at 50 mT. Remove the vial holder from the lyophilizer, and screw the vials with aluminum seals. Table 26 Step number step Temperature or temperature change rate Time (min) Pressure ( mTorr ) 1 Load/balance 5℃ 60 2 Change from 5℃ to 0℃ 0.5℃/min 10 3 maintain 0℃ 60 4 Change from 0℃ to -45℃ 1℃/min 45 5 maintain -45℃ 120 6 Gasp -45℃ 60 50 7 Change from -45℃ to -15℃ 0.3°C/min 100 50 8 maintain -15℃ 2710 50 9 Change from -15 ℃ to 35 ℃ 0.2℃/min 250 50 10 maintain 35°C 420 50 11 Change from 35℃ to 5℃ 0.5℃/min 60 50 12 maintain Keep 5℃ 60 50 Experimental design of freeze-dried formulations

For each fill volume configuration, in addition to 5 placebo vials, 5 product vials are filled. For each fill volume at T=0, test 2 vials from each active product and placebo (1 vial is used to test residual moisture, and 1 vial is reconditioned and tested using the analysis described in the test summary). In addition, two vials from each active and placebo were probed for temperature monitoring during the freeze-drying process.

The standard formulation used in this test is 12.8 mg/mL, containing 5.0% sucrose, 0.02% Tween 20 pH 6.0, which is the starting material of AGS-22M6E. result

Lyophilization cycle analysis : The total conservative cycle time is 2.6 days, which is significantly shorter than the established cycle time of 4.7 days for a filling volume of 5 mL (see section 6.3 above). However, the cycle time of 2.6 days in the test of the present invention does not indicate an optimized cycle time for any filling volume. After careful evaluation of the individual readings related to the cycle, the optimal cycle time for any fill volume can be estimated. Since dew point monitoring responds to the release of water from two fill volumes, it is not used in this test to estimate the better drying time for a 3.0 mL or 1.5 mL fill volume. Similarly, in this case, the Pirani vacuum gauge (although it is usually an excellent indicator of the end point of the initial drying time) only provides guidance because it is too responsive to the sublimation of ice from all vials on the shelf . In fact, for the purpose of this test, monitoring the product temperature provides the most reliable tool for evaluating the drying time of each individual filling volume. In this experiment, the thermocouple probe was placed in both the active vial and the placebo vial, and for the two fill volumes, an excellent agreement was observed between the product temperature profiles of the active and the placebo.

Under the condition that the product temperature provides an accurate measurement of the drying time, the comparison of the two filling volume data (not shown in this data) shows that the 1.5 mL filling volume dries in approximately 1.3 days, which is much faster than 3.0 The mL fill volume dries in approximately 1.8 days.

Cake appearance : All the cakes are complete cakes, with very small shrinkage and a shiny surface. All of them maintain a sound structure. In most vials, there are no cracks in the cake. When the vial is reversed, the cake will not still adhere to the vial, but will tumble perfectly in the vial. In some vials, fine cracks were observed around the edge of the bumpy circle (where the cake was attached to the vial). For the two filling volumes, there was no difference between the cake formation of the active and the placebo. The appearance of the cake is also equivalent to the cake appearance recorded in the 5 mL filling volume of the freeze-dried product in the test in Section 6.3.

A280 analysis : Before filling and freeze-drying, check the protein concentration of the formulation, repeat twice, and find that it is close to the target concentration of 10 mg/mL. After reconstitution with 2.8 mL and 1.4 mL WFI, respectively, the protein concentrations of the 3.0 mL and 1.5 mL filled formulations were also close to their protein concentrations before lyophilization (see the table below). Table 27 sample Dilution factor (df) A330nm A280nm [Protein] mg/mL Average (mg/mL) Before freeze-drying 20 0.00026 0.73176 10.06 10.1 0.00000 0.74046 10.19 F4 10 mg/mL, T=0, 3 mL 20 0.00126 0.82283 11.30 11.3 0.00000 0.81551 11.22 F4 10 mg/mL, T=0, 1.5 mL 20 0.00429 0.78214 10.70 10.7

Residual moisture, recovery time, A330 , weight molar osmolality and visual appearance : After the completion of lyophilization, a vial of each fill volume from the active vial and the placebo vial was allocated for the residual moisture test. As shown in Table 28 below, the residual moisture of the actives ranges from 0.18% (for 3.0 mL filling) to 0.29% (for 1.5 mL filling). For the two filling volumes, the residual moisture of the placebo was slightly higher, at 0.34%. For all the vials tested, the recovery time was less than 1 min, where the time recorded for the 3.0 mL filling volume (average 36 s) was slightly higher compared to the 1.5 mL filling volume sample (average 20 s). No significant difference was observed between the recovery time of the active vial and the placebo vial. Similarly, the visual appearance of all reconstituted samples (both active and placebo) was reported as clear and colorless and particle-free. Table 28 condition Lyophilized fill volume Recovery time^ (sec) Turbidity (A330) Weight molar osmotic concentration (mOsm/kg) 1 Residual moisture %* Active matter Placebo Active matter Placebo Active matter Placebo Active matter Placebo T=0 3.0 38 35 0.0645 0.0139 194 186 0.18 0.34 1.5 twenty two 18 0.0555 0.0097 190 189 0.29 0.34 Before freeze-drying 0.0610 0.0142 190 181 0.0492 0.0089

The turbidity (A330) measurements of the active vials and the placebo vials are also shown in the table above. As shown, for both samples before and after lyophilization, the A330 value of the active vial was slightly higher than that of the placebo, confirming the previous results discussed in section 6.3, which showed that AGS-22M6E contributed to the turbidity of the formulation.

Before filling and after recovery, test the weight molar osmolality of the prepared sample and buffer, and repeat twice. Table 28 above summarizes this data and shows that there is no significant difference in osmolality by weight before lyophilization or after reconstitution. It is also equivalent to the weight molar osmolality determined for the 5.0 mL filled configuration sample in section 6.3.

Figure 4 shows the SDS-PAGE analysis (reduced and non-reduced) of each filling volume compared to the standard formulation. The results show that changing the fill volume has no effect on the SDS-PAGE profile.

Table 29 below summarizes the SEC-HPLC data, including the percentages of HMW peak, main peak, and LMW peak of samples freeze-dried at each fill volume. Regardless of the filling volume evaluated, the lyophilized samples behaved similarly and there was no difference in the measured main peak area before and after lyophilization. No change was observed for any filling volume, and the overview was equivalent to the lyophilized 5 mL filling volume in the experiment described in Section 6.3. Table 29 Peak percentage (%) Peak area (mAu) Sample name Main peak retention time Front peak Main peak Back peak Front peak Main peak Back peak total Reference standard 19.8 1.4 95.2 3.4 429 29155 1045 30629 F4, active substance, lyophilized, filled with 3.0 mL 19.8 1.3 95.4 3.3 481 34059 1174 35714 F4, active substance, lyophilized, 1.5 mL filled 19.8 1.4 95.1 3.5 487 32359 1198 34043

Based on the foregoing, it should be understood that although specific embodiments have been described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope provided herein. All references mentioned above are incorporated herein by reference in their entirety. 7. Sequence Listing

This manual is submitted together with a computer-readable copy (CRF) of the sequence listing. The CRF titled "14369-244-228_SEQ_LISTING.txt" created on October 11, 2019 with a size of 39,693 bytes is incorporated herein by reference in its entirety.

Figures 1A , 1B , 1C and 1D depict the results of the SDS-PAGE analysis of the 14-day stability test of formulations F1-F14 at 40°C. Figure 1E depicts an overview of the PR-HPLC test described in section 6.1. Figures 1F , 1G and 1H depict the results of SE-HPLC analysis of formulations F1-F14 at 40°C. Figure 2A depicts the results of a shake study of formulations F4, F9 and F14 at T0. Figure 2B depicts the SDS-PAGE results of the cyclic freeze-thaw test of formulations F4, F9 and F14. Figure 2C depicts the total cumulative counts per milliliter for formulations F4, F9 and F14, as measured by HIAC. Figure 3A depicts the results of residual moisture analysis of formulations F4, F9 and F14. Figure 3B depicts the results of A280 (concentration) in a 12-week simultaneous BDS and DP formulation test. Figure 3C depicts the results of A330 (turbidity) in a 12-week simultaneous BDS and DP formulation test. Figure 3D depicts the results of SDS-PAGE analysis of BDS (before lyophilization) at TO. Figure 3E depicts the results of SDS-PAGE analysis of BDS stored at -70°C or 2-8°C for 12 weeks. Figure 3F depicts the results of SDS-PAGE analysis of DP after lyophilization and restoration at T0. Figure 3G depicts the results of SDS-PAGE analysis of DP (after lyophilization and reconstitution) stored at 25°C or 40°C for 12 weeks. Figure 3H depicts the results of SDS-PAGE analysis of DP (after lyophilization and reconstitution) stored at 2-8°C for 12 weeks. Figure 3I depicts the AGS-22M6E BDS stored at 2-8°C and -70°C for 12 weeks, and the freeze-drying stored at 2-8°C, 25°C/60% RH and 40°C/75% RH for 12 weeks SE-HPLC analysis result of AGS-22M6E. Figure 4 depicts the results of SDS-PAGE analysis of the freeze-dried formulation of F4 at a fill volume of 3.0 and 1.5 mL. Figure 5A depicts the nucleotide and amino acid sequences of the 191P4D12 protein. Figure 5B depicts the nucleotide and amino acid sequences of the heavy and light chains of Ha22-2(2.4)6.1. Figure 5C depicts the amino acid sequences of the heavy and light chains of Ha22-2(2.4)6.1.

Claims (114)

A pharmaceutical composition comprising (a) An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof that binds to 191P4D12 bound to one or more monomethyl auristatin E (MMAE) units, wherein the antibody or antigen-binding fragment thereof Comprises a heavy chain variable region containing a complementarity determining region (CDR), which includes the amino acid sequence of the CDR of the heavy chain variable region set forth in SEQ ID NO: 7; and the light chain containing CDR Variable regions, the CDRs comprising the amino acid sequence of the CDR of the light chain variable region set forth in SEQ ID NO: 8; and (b) A pharmaceutically acceptable excipient, which includes at least one of L-histidine, polysorbate-20 (TWEEN-20), and trehalose dihydrate and sucrose. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: CDR H1, which comprises the amino acid sequence of SEQ ID NO: 9; CDR H2, which comprises the amino acid sequence of SEQ ID NO: 10 CDR H3, which includes the amino acid sequence of SEQ ID NO: 11; CDR L1, which includes the amino acid sequence of SEQ ID NO: 12; CDR L2, which includes the amino acid sequence of SEQ ID NO: 13; and CDR L3, which includes the amino acid sequence of SEQ ID NO:14. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the 20th amino acid (glutamic acid) to the 136th amino acid of SEQ ID NO: 7 Amino acid sequence within the range of base acid (serine); and the light chain variable region, which is contained in SEQ ID NO: 8 from the 23rd amino acid (aspartic acid) to the 130th amino acid (Arginine) Amino acid sequence within the range. The pharmaceutical composition of claim 1, wherein the antibody comprises: a heavy chain, which is included in the range of the 20th amino acid (glutamic acid) to the 466th amino acid (lysine) of SEQ ID NO: 7 The amino acid sequence within; and the light chain, which includes the amino acid within the range of the 23rd amino acid (aspartic acid) to the 236th amino acid (cysteine) of SEQ ID NO: 8 Acid sequence. The pharmaceutical composition according to any one of claims 1 to 4, wherein the antigen-binding fragment is a Fab, F(ab') ₂ , Fv or scFv fragment. The pharmaceutical composition according to any one of claims 1 to 5, wherein the antibody is a fully human antibody. The pharmaceutical composition according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is produced recombinantly. The pharmaceutical composition according to any one of claims 1 to 7, wherein the antibody-drug conjugate has the following structure:

Wherein L-represents the antibody or its antigen-binding fragment, and p is 1-10. The pharmaceutical composition of claim 8, wherein p is 2-8. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding fragment is connected to each monomethyl auristatin E (MMAE) unit via a linker. The pharmaceutical composition of claim 10, wherein the linker is an enzyme-cleavable linker, and wherein the linker forms a bond with the sulfur atom of the antibody or antigen-binding fragment thereof. The pharmaceutical composition of claim 10, wherein the linker has the formula -A _a -W _w -Y _y -, wherein -A- is an extension unit, a is 0 or 1; -W- is an amino acid unit , W is an integer in the range of 0-12; and -Y- is a spacer unit, y is 0, 1 or 2. The pharmaceutical composition of claim 12, wherein the extension unit has the structure of the following formula (1); the amino acid unit is valine citrulline; and the spacer unit is a PAB group, which comprises the following formula (2) Structure:

Formula 1)

Formula (2). The pharmaceutical composition of claim 12, wherein the extension unit forms a bond with the sulfur atom of the antibody or antigen-binding fragment thereof; and wherein the spacer unit is connected to MMAE via a carbamate group. The pharmaceutical composition of claim 1, wherein each antibody or antigen-binding fragment thereof of the antibody-drug conjugate contains 1 to 10 MMAE units. The pharmaceutical composition of claim 15, wherein each antibody or antigen-binding fragment thereof of the antibody-drug conjugate contains 2 to 8 MMAE units. The pharmaceutical composition according to any one of claims 1 to 16, which comprises the antibody drug conjugate at a concentration of 1 to 20 mg/mL. The pharmaceutical composition of claim 17, which contains the antibody-drug conjugate at a concentration of 5 to 15 mg/mL. The pharmaceutical composition of claim 17, which comprises the antibody drug conjugate at a concentration of 8 to 12 mg/mL. The pharmaceutical composition of claim 17, which comprises the antibody-drug conjugate at a concentration of about 10 mg/mL. The pharmaceutical composition according to any one of claims 1 to 20, wherein the content of the L-histidine is in the range of 5 to 50 mM. The pharmaceutical composition according to any one of claims 1 to 20, wherein the content of the L-histidine is in the range of 10 to 40 mM. The pharmaceutical composition according to any one of claims 1 to 20, wherein the content of the L-histidine is in the range of 15 to 35 mM. The pharmaceutical composition according to any one of claims 1 to 20, wherein the content of the L-histidine is in the range of 15 to 30 mM. The pharmaceutical composition according to any one of claims 1 to 20, wherein the content of the L-histidine is in the range of 15 to 25 mM. The pharmaceutical composition according to any one of claims 1 to 20, wherein the content of the L-histidine is about 20 mM. The pharmaceutical composition according to any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of 0.001 to 0.1% (v/v). The pharmaceutical composition according to any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of 0.0025 to 0.075% (v/v). The pharmaceutical composition according to any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of 0.005 to 0.05% (v/v). The pharmaceutical composition according to any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of 0.01 to 0.03% (v/v). The pharmaceutical composition according to any one of claims 1 to 26, wherein the concentration of TWEEN-20 is in the range of about 0.02% (v/v). The pharmaceutical composition according to any one of claims 1 to 31, wherein the pharmaceutical composition comprises trehalose dihydrate. The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is in the range of 1 to 20% (w/v). The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is in the range of 2 to 15% (w/v). The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is in the range of 3 to 10% (w/v). The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is in the range of 4 to 6% (w/v). The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is about 5.5% (w/v). The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is in the range of 50 mM to 300 mM. The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is in the range of 75 mM to 250 mM. The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is in the range of 100 mM to 200 mM. The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is in the range of 130 mM to 150 mM. The pharmaceutical composition of claim 32, wherein the content of the trehalose dihydrate is about 146 mM. The pharmaceutical composition according to any one of claims 1 to 31, wherein the pharmaceutical composition comprises sucrose. The pharmaceutical composition of claim 43, wherein the content of the sucrose is in the range of 1 to 20% (w/v). The pharmaceutical composition of claim 43, wherein the content of the sucrose is in the range of 2 to 15% (w/v). The pharmaceutical composition of claim 43, wherein the content of the sucrose is in the range of 3 to 10% (w/v). The pharmaceutical composition of claim 43, wherein the content of the sucrose is in the range of 4 to 6% (w/v). The pharmaceutical composition of claim 43, wherein the content of the sucrose is about 5.5% (w/v). The pharmaceutical composition of claim 43, wherein the content of the sucrose is in the range of 50 mM to 300 mM. The pharmaceutical composition of claim 43, wherein the content of the sucrose is in the range of 75 mM to 250 mM. The pharmaceutical composition of claim 43, wherein the content of the sucrose is in the range of 100 mM to 200 mM. The pharmaceutical composition of claim 43, wherein the content of the sucrose is in the range of 130 mM to 150 mM. The pharmaceutical composition of claim 43, wherein the content of the sucrose is about 146 mM. The pharmaceutical composition according to any one of claims 1 to 53, wherein the pharmaceutical composition has a pH in the range of 5.5 to 6.5. The pharmaceutical composition according to any one of claims 1 to 53, wherein the pharmaceutical composition has a pH in the range of 5.7 to 6.3. The pharmaceutical composition according to any one of claims 1 to 53, wherein the pharmaceutical composition has a pH of about 6.0. The pharmaceutical composition according to any one of claims 54 to 56, wherein the pH is measured at room temperature. The pharmaceutical composition according to any one of claims 54 to 56, wherein the pH is measured at 15°C to 27°C. The pharmaceutical composition according to any one of claims 54 to 56, wherein the pH is measured at 4°C. The pharmaceutical composition according to any one of claims 54 to 56, wherein the pH is measured at 25°C. The pharmaceutical composition according to any one of claims 1 to 60, which comprises hydrochloric acid (HCl). The pharmaceutical composition according to any one of claims 1 to 60, wherein the pH is adjusted using HCl. The pharmaceutical composition according to any one of claims 1 to 60, which comprises succinic acid. The pharmaceutical composition according to any one of claims 1 to 60, wherein the pH is adjusted using succinic acid. The pharmaceutical composition according to any one of claims 1 to 16, which comprises about 20 mM L-histidine, about 0.02% (w/v) TWEEN-20, and about 5.5% (w/v) seaweed dihydrate At least one of sugar or about 5% (w/v) sucrose. The pharmaceutical composition of claim 65, which further comprises HCl or succinic acid. The pharmaceutical composition of claim 65 or claim 66, wherein the pH at room temperature is 6.0. The pharmaceutical composition of claim 65 or claim 66, wherein the pH at 25°C is 6.0. A pharmaceutical composition comprising (a) an antibody-drug conjugate, which comprises the following structure:

Wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mM L-histidine, about 0.02% (w/v ) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and HCl; wherein the pH at 25°C is about 6.0. The pharmaceutical composition of claim 69, wherein the concentration of the antibody-drug conjugate is about 10 mg/mL. A pharmaceutical composition comprising (a) an antibody-drug conjugate, which comprises the following structure:

Wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mM L-histidine, about 0.02% (w/v ) TWEEN-20, about 5.5% (w/v) trehalose dihydrate and succinic acid; wherein the pH at 25°C is about 6.0. The pharmaceutical composition of claim 71, wherein the concentration of the antibody-drug conjugate is about 10 mg/mL. A pharmaceutical composition comprising (a) an antibody-drug conjugate, which comprises the following structure:

Wherein L- represents an antibody or antigen-binding fragment thereof, and p is 1 to 10; and (b) a pharmaceutically acceptable excipient, which contains about 20 mM L-histidine, about 0.02% (w/v ) TWEEN-20, about 5.0% (w/v) sucrose and HCl; wherein the pH at 25°C is about 6.0. The pharmaceutical composition of claim 73, wherein the concentration of the antibody-drug conjugate is about 10 mg/mL. The pharmaceutical composition according to any one of claims 1 to 74, wherein the pharmaceutical composition is in a liquid form. The pharmaceutical composition according to any one of claims 1 to 74, wherein the pharmaceutical composition has been lyophilized. A freeze-dried composition, which is prepared by freeze-drying the pharmaceutical composition according to any one of claims 1 to 74. The pharmaceutical composition according to any one of claims 1 to 74, wherein the pharmaceutical composition is stored at -80°C, 4°C, 25°C or 37°C. A method for preventing or treating a disease or condition in an individual, which comprises administering to the individual an effective amount of the pharmaceutical composition according to any one of claims 1 to 78. The method of claim 79, wherein the individual is a human individual. The method of claim 80, wherein the cancer is colon cancer, pancreatic cancer, ovarian cancer, lung cancer, bladder cancer, urothelial cancer, breast cancer, esophageal cancer, head cancer or neck cancer. The method of claim 81, wherein the cancer is colon cancer. The method of claim 81, wherein the cancer is pancreatic cancer. The method of claim 81, wherein the cancer is ovarian cancer. The method of claim 81, wherein the cancer is lung cancer, and the lung cancer is non-small cell lung cancer as appropriate. The method of claim 81, wherein the cancer is bladder cancer or urothelial cancer. The method of claim 86, wherein the bladder cancer is advanced bladder cancer or advanced urothelial cancer. The method of claim 86, wherein the bladder cancer is metastatic bladder cancer or metastatic urothelial cancer. The method of claim 81, wherein the cancer is breast cancer. The method of claim 81, wherein the cancer is esophageal cancer. The method of claim 81, wherein the cancer is head cancer. The method of claim 81, wherein the cancer is neck cancer. The method of claim 80, wherein the cancer has tumor cells expressing 191P4D12. The method of any one of claims 79 to 93, further comprising administering a second therapeutic agent to the individual. The method of claim 94, wherein the second therapeutic agent is an immune checkpoint inhibitor. The method of claim 95, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. The method of claim 96, wherein the immune checkpoint inhibitor is a PD-1 inhibitor. The method of claim 97, wherein the PD-1 inhibitor is nivolumab. The method of claim 96, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor. The method according to claim 99, wherein the PD-L1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. The method according to any one of claims 79 to 100, wherein the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of 1 to 10 mg/kg body weight. The method of claim 101, wherein the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of 1 to 5 mg/kg body weight. The method of claim 101, wherein the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of 1 to 2.5 mg/kg body weight of the individual. The method of claim 101, wherein the antibody-drug conjugate formulated in the pharmaceutical composition is administered at a dose of 1 to 1.25 mg/kg body weight of the individual. The method of claim 101, wherein the antibody drug conjugate formulated in the pharmaceutical composition is administered at a dose of about 1 mg/kg body weight of the individual. The method of claim 101, wherein the antibody drug conjugate formulated in the pharmaceutical composition is administered at a dose of about 1.25 mg/kg body weight of the individual. The method according to any one of claims 101 to 106, wherein the antibody drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion. The method of claim 107, wherein the antibody-drug conjugate formulated in the pharmaceutical composition is administered twice every three weeks by intravenous (IV) injection or infusion for about 30 minutes. The method of claim 108, wherein the antibody-drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion for about 30 minutes on the first and eighth days of every three-week cycle. The method of claim 109, which further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion on the first day of every three-week cycle. The method of claim 110, wherein the immune checkpoint inhibitor is administered in an amount of about 100 mg to about 1500 mg over about 30 minutes or 60 minutes. The method of claim 107, wherein the antibody-drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection or infusion for about 30 minutes three times every four weeks. The method of claim 112, wherein the antibody-drug conjugate formulated in the pharmaceutical composition is administered by intravenous (IV) injection for about 30 minutes on the first 1, 8 and 15 days of every four-week cycle. The method of claim 113, which further comprises administering an immune checkpoint inhibitor by intravenous (IV) injection or infusion.

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